Chimeric chains that coding for proteins that induce effects directed against viruses

ABSTRACT

The present invention is related to the obtaining of chimeric chains coding for proteins capable of inducing, in the recipient, a serotype-specific and protective humoral immune response against the infection by the Dengue virus, thus eliminating the effects of the serotype-nonespecific viral immunoenhancement that causes hemorrhagies and clinical complications described for this kind of pathology. These chimeric chains of nucleic acids are composed by the specific combination of fragments belonging to the gene of a mutated protein from  Neisseria meningitidis  with dehydrogenase activity and fragments that codify for a region of the envelope (E) protein from the Dengue virus which, when inserted to an expression vector, give rise to chimeric proteins with particular properties. The resultant chimeric molecules from this invention are applicable to the pharmaceutical industry for the obtaining of vaccine preparations and diagnostic means of high serotype-specificity to be used in humans.

FIELD OF THE INVENTION

[0001] The present invention is related with the field of the biotechnology and the pharmaceutical industry, in particular with the obtaining of chimeric nucleotide chains which, when introduced into an expression vector, give rise to proteins able to elicit a serotype-specific humoral immune response and protection against the Dengue virus infection; quoted from now on as DEN, avoiding the effects of the serotype-specific viral immunoamplification, causing hemorrhages and clinical complications described in this type of pathology.

PREVIOUS TECHNIQUE

[0002] The Dengue virus (DEN), is a coated virus whose lipid membrane contains two of its three structural proteins: the envelope protein (E) and the membrane protein (M). The E protein covers an icosaedric nucleocapsid composed by the third of its structural proteins, the core protein. This virus belongs to the Flaviviridae family and four different serotypes exist. Its transmission to the man is carried out through the mosquito Aedes aegypti that belongs to the Stegomia family. The disease produced in the human for this virus was considered as benign and was described as Dengue Fever or Classical Dengue (DF) until the appearance of a more serious modality and sometimes lethal, characterized by hemorrhagic fever and shock, denominated: Hemorrhagic Dengue Fever and Dengue Shock Syndrome (HDF/DSS) (Hammon WMc. New hemorrhagic fever in children in the Philippines and Thailand. Trans Assoc Physicians 1960; 73: 140-155). Several epidemiological studies have been carried out evidencing as a risk factor the sequential infection of two different viral serotypes (Kouri G P, Guzman M G, Brave J R. Why dengue hemorrhagic fever in Cuba) 2. An integral analysis. Trans Roy Soc Trop Med Hyg 1987; 72: 821-823). This phenomenon is explained by the immuno-enhancement, which is based on an increase of the viral infection by increasing the entrance of the virus-antibody complex to the cell through the Fc receptors of the target cell (monocytes) (haistead SB. Pathogenesis of dengue: challenges to molecular biology. Science 1988; 239: 476-481).

[0003] Different technologies have been developed to produce live attenuated vaccines, but at present there exist multiple unsolved issues on the possible benefits of these vaccines, since they could revert to the virulence, viral interference and inter-genomic recombination. Alternately, recombinant antigens can be obtained as possible components of a subunit vaccine (Feighny, R., Borrous, J. and Putnak R. Dengue type-2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge. Am. J. Trop. Med. Hyg. 1994. 50(3). 322-328; Deubel, V., Staropoli, I., Megret, F., et al. Affinity-purified dengue-2 virus envelope glycoprotein induces neutralizing antibodies and protective immunity in mice. Vaccine. 1997. 15, 1946-1954).

[0004] The main antigen of the virus is the envelope protein DENe. This protein is the major component of the viral surface and is thought to mediate the binding of the virus to the cellular receptor (A Heinz F X, Berge R, Tuma W et al. A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies. Virology. 1983; 126: 525). This protein has structural homology with that of the tick borne encephalitis virus (TBE) (Rey, F. A., Heinz, F. X., Mandl, C., et al. The envelope glycoprotein from tick borne encephalitis virus at 2 A° resolution. Nature 1995; 375: 291-298) and it is also structurally conserved among serotypes.

[0005] The insect cells constitute one of the systems most used for the expression of diverse heterologous genes that employ the baculovirus system as vectors. These vectors have been used for the expression of several combinations of structural and nonstructural proteins of the Encephalitis Japanese virus (JEV), DEN-1, DEN-2 and DEN4, (Matsuura Y, Miyamoto M, Soto T et al. Characterization of japanese encephalitis virus envelope protein expressed by recombinant baculoviruses. Virology 1989; 173: 677-682; Deubel V, Bordier M, Megret F et al. Processing, secretion and immunoreactivity of carboxy terminally truncated dengue-2 envelope proteins expressed in insect cell by recombinant baculoviruses. Virology 1991; 180: 440-447; Putnak R, Feighny R, Burrous J et al. Dengue 1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicit virus neutralization antibodies in mice and protects them from virus challenge. Am J Trop Med Hyg 1991; 45: 159-167; Feighny R, Burrous J, Putnak R. Dengue type 2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge. Am J Trop Med Hyg 1994; 50: 322-328). Another system used has been the cells of Drosophila melanogaster expressing different variants of the E protein (PCT/US96/07627). In spite of obtaining an appropriate functional response, upon using the proteins expressed in these systems, they imply a high cost for the development of scale-up production processes; therefore, the expression in yeast has been an alternative to produce recombinant structural proteins of flavivirus. However, in the case of the DENe protein, expressed in Pichia pastoris (PCT/US96/07627; Sugrue R. J., Fu H., Howe J., Chan Y. Expression of the Dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles. J. Virol. 1997. 78, 1861-1866), the levels of expression are low, either secreted or intracellularly, hindering the purification process.

[0006] In parallel, several variants of the DENe protein have been obtained in bacteria. One of them was the C-terminal portion of the E protein of the JEV fused to a protein of the Tryptophan metabolism (TrpE) of E. coli. This protein was produced as inclusion bodies and was recognized by neutralizing monoclonal antibodies (Mabs) using immunodetection techniques. However, pure preparations of this protein were unable to develop neutralizing antibodies and to protect against viral challenge (Mason P. W., Zogel M. V., Semproni A. R., et al. The antigenic structure of dengue type I virus envelope and NS1 protein expressed in E. coli. J Gen Virol. 1990. 71: 2107-2114). In addition, another construction was made (Srivastava A. K., Morita K., Matsuo S., et al. Japanese encephalitis virus fusion protein with protein A expressed in E. coli confers protection in mice. Microbiol Immunol. 1991. 35: 863-870), that contains the protein A of Staphylococcus aurius fused to the C-terminal fragment of the E protein, followed by the N-terminal segment of the nonstructural protein of the JEV, the NS1. In this case the fused protein was soluble, facilitating its purification by affinity chromatography. Upon immunizing mice with this pure protein high neutralizing antibodies titers were obtained, which also inhibited haemagglutination and protected against the viral challenge with the JEV. Similar results were obtained using the DENe region of the DEN-2 fused to the protein A of S. aureus (Srivastava A. K., Putnak R. J., Warren R. L., Hoke C. H. Mice immunized with a dengue type 2 virus E and NS1 fusion protein made in Escherichia coli are protected against lethal dengue virus infection. Vaccine. 1995. 13: 1251-1258); however, it is not possible to use these preparations in humans due to the presence of the protein A, which has shown a high affinity for the human immunoglobulin G (IgG).

[0007] Finally, it has been reported a fusion protein that contains the B domain of the DENe protein of DEN-2 and the maltose binding protein (MBP) of E. coli (Simmons M., Nelson W. M., Wu S. J., Hayes C. G. Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice. Am J Trop Med Hyg. 1998. 58: 655-662) denominated MBP-DomB. This protein variant was immunogenic in mice and elicited neutralizing antibodies.

[0008] In our case, the subject of this invention relies on the obtaining of chimeric sequences, as for instance, in the first case, the sequence coding for a region of the DENe protein linked to the N-terminal fragment of a mutated protein with dehydrogenase activity (MDH) from Neisseria meningitidis; in the second case, the sequence coding for a region of the DENe protein linked to the entire gene of the MDH protein in two different positions, and in the third case, the chimeric sequences are formed by two fragments of the DENe protein from two different viral serotypes fused to the same gene coding for the MDH protein. These chimeric chains when inserted into a suitable vector, give rise to insoluble chimeric proteins within the bacterium's cytoplasm. These proteins are then capable to elicit high levels of neutralizing antibodies against DEN, inhibitors of the viral hemagglutination and to protect immunized mice against viral challenge.

[0009] With regards to the insolubility of the aforementioned proteins, an easy scale-up folding process was achieved in vitro, as well as the expression and purification processes which were superior to those used by Simmons et al, 1998. On the other hand, the serotype specificity of the antibodies is demonstrated, generated by immunization of mice with these proteins, at the level of neutralization, inhibition of hemaglutination and ELISA, using doses lower than those employed by Simmons et al, 1998. This fact constitutes the first report on the expression of insoluble DENe proteins in E. coli capable of stimulating a functionl immune response.

[0010] In addition, considering the results obtained with the dimeric variant, it is possible to generate serotype-specific antibodies with the same molecule for two different viral serotypes, capable of neutralizing viral infection and protect mice against viral challenge. Concerning the MDH protein, a search for homology with other sequences was done in the EMBL data base, revealing that the first 110 amino acids are highly similar to the lipoic binding domain region and the flexible hinge of the dihydrolipoamide acetyltransferase (E2 enzyme of the pyruvate dehydrogenase complex and α-cetoglutarate dehydrogenase), and the rest of the protein is highly similar to the lipoamide dehydrogenase (LPDH), enzyme E3 of said complexes (Stephens, P. E; H. M. Darlinson, and J. R. Guest., 1983. The Pyruvate dehydrogenase complex of E. coli. Eur. J. Biochem. 133:155-162.

[0011] On the other hand, it was also found that patients with Primary Biliary Cirrhosis (PBC) produced anti-mitochondrial autoantibodies, specific for the lipoic acid binding site, common among these proteins (Gershwin M E, Mackay I R, Sturgess A, Coppel R L. Identification and specificity of a cDNA encoding the 70 KDa mitochondrial antigen recognized in primary biliary cirrhosis. J Immunol 1987;138:3525-31). Therefore, we decided to mutate this region within the protein to avoid any autoimmune response when immunized in humans as chimeric proteins. The mutated MDH protein of our invention was used in a Phase I clinical trial and showed to be safe and immunogenic in humans, and also was not recognized by sera of patients with PBC (Perez, A., F. Dickinson, Z. Cinza, A. Ruiz, T. Serrano, J. Sosa, S. González, Y. Gutiérrez, C. Nazábal, O. Gutiérrez, D. Guzmán, M. Diaz, M. Delgado, E. Caballero, G. Sardiñas, A. Alvarez, A. Martin, G. Guillén, R. Silva. Safety and preliminary immunogenicity of the recombinant outer membrane protein of Neisseria meningitidis in human volunteers. Biotech. Appl. Biochem. 34: 121-125). However, the possible use of the MBP in humans has not been demonstrated yet (Simmons M., Nelson W. M., Wu S. J., Hayes C. G. Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice. Am J Trop Med Hyg. 1998. 58: 655-662).

DETAILED DESCRIPTION OF THE INVENTION

[0012] In this invention is described the obtaining of chimeric nucleotide chains that when introduced into an expression vector, give rise to chimeric proteins capable of inducing a serotype-specific humoral immune response and protecting against the infection by Dengue virus, as for instance, the sequence coding for a region of the DENe protein from each one of the viral serotypes of the Dengue virus, linked to the N-terminal fragment of a mutated protein with dehydrogenase activity (MDH) from Neisseria meningitidis; in the second case, the sequence coding for a region of the DENe protein linked to the entire gene of the MDH protein in two different positions: within one site of the sequence coding for the structural domain of the MDH protein (lipoic acid binding domain and the 3′ end of the gen), and in the third case, the chimeric sequences are formed by two fragments of the DENe protein from two different viral serotypes, DEN-2 and DEN-4, in two different positions of the MDH gen: one within a particular site of the sequence coding for the lipoic acid binding domain (serotype 4) and the other in the 3′ end of the MDH gen (serotype 2). This was called a dimeric construct.

[0013] This chimeric proteins were obtained insoluble within the bacterium's cytoplasm. A purification process by immobilazed metal affinity chromatography (IMAC) was done which led to obtain pure proteins for immunogenicity studies.

[0014] When antigenicity results were analyzed a strong recognition of all the recombinant chimeric proteins for the hyperimmune ascitic liquids (HMAF) anti-DEN was demonstrated, being higher for the case of the fusion to the entire MDH gene, which evidences a positive effect on the folding of the region from the DENe protein given by the MDH. In the cases where the serotype 2 was used, all recombinant proteins obtained were recognized by a serotype-specific neutralizing antibody (3H5), being also higher for the case of the fusion to the entire MDH gene, as well as in the dimeric protein. It was also observed that the recognition for the HMAF from the homologous serotype in each case was significantly higher than the recognition for the HMAF from the heterologous serotypes, evidencing the exposure of serotype-specific epitopes and permitting thus its use as a diagnostic mean for Dengue and serotyping.

[0015] When all the recombinant chimeric proteins were immunized in mice a neutralizing and protective response was obtained. Highest neutralizing titers were obtained with the sequences fused to the entire gene of the MDH and with the dimeric protein, independently of the position of the fragment from the DENe protein. This showed an immunopotentiator effect of the immune response mediated by the MDH that can be explained by the influence in the folding of the DENe protein reflected in the antigenicity results obtained. It was also demonstrated for the first time, and contrary to the previous state of the art, that the insolubility of these proteins do not affect the capacity of generating a suitable immune response.

[0016] The immune response produced in all the cases was serotype-specific (antibodies against the immunized homologous serotype) in the viral neutralization, the hemoagglutination inhibition and ELISA. The generation of serotype-specific antibodies means that they are not capable to recognize antigenic determinants from virus of heterologous serotypes that favour the immunoenhacement phenomenum. This characteristic is of great importance for the development of a vaccine candidate against the Dengue virus since the recognition of antibodies to heterologous serotypes could be one of the causes for the Hemorrhagic Dengue Fever (HDF).

[0017] Besides it was showed the induction of antibodies against two viral serotypes after immunization with just one of the chimeric proteins, which permits the formulation of a vaccine candidate against the four serotypes, using only two of our available recombinant chimeric proteins.

[0018] The obtaining of the mutant MDH protein consisted of the elimination of the lipoic acid binding site in the sequence ETDKAT, based on the covalent binding of this fatty acid with the epsilon-amine groups of lysine (K) (Tuaillon N, Andre C, Briand J P et al. A lipoyl synthetic octadecapeptide of dihydrolipoamide acetyltransferase specifically recognized by anti-M2 autoantibodies in Primary Biliary Cirrhosis. J Immunol 1992; 148:445-50).

[0019] The mutagenesis was done by using PCR with a pair of primers to amplify the N-terminal region (from the start codon of the lpdA gene until the lipoic acid binding site, 135 bp) and the C-terminal of the protein (from the lipoic acid binding site until the 3′ end of the gene); thus, being eliminated the possibility to generate autoimmune reactions, as demonstrated in the human clinical trials.

[0020] Deposit of the Biological Material

[0021] Plasmids PLL1, PLL2, PLL3, PLH1, PLH2, PLH3, PAZ1, PAZ2, PAZ3, PID1, PID2 and PID3 were deposited according to the Budapest Treaty in the Belgian Coordinated collection of Microorganism—BCCM™, LMBP-COLLECTION, and with the date and under the access numbers

DESCRIPTION OF FIGURES

[0022]FIG. 1. Cloning strategy of the E2 fragment to obtain PLL1.

[0023] DENe2: Fragment of the envelope protein of DEN-2.

[0024] N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.

[0025]FIG. 2. Cloning strategy of the E2 fragment to obtain PLL2.

[0026] DENe2: Fragment of the envelope protein of DEN-2.

[0027] MDH: dehydrogenase mutant.

[0028]FIG. 3. Cloning strategy of the E2 fragment to obtain PLL3.

[0029] DENe2: Fragment of the envelope protein of DEN-2.

[0030] MDH: dehydrogenase mutant.

[0031]FIG. 4. Cloning strategy of the E1 fragment to obtain PLH1.

[0032] DENe1: Fragment of the envelope protein of DEN-1.

[0033] N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.

[0034]FIG. 5. Cloning strategy of the E1 fragment to obtain PLH2.

[0035] DENe1: Fragment of the envelope protein of DEN-1.

[0036] N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.

[0037]FIG. 6. Cloning strategy of the E1 fragment to obtain PLH3.

[0038] DENe1: Fragment of the envelope protein of DEN-1.

[0039] MDH: dehydrogenase mutant.

[0040]FIG. 7. Cloning strategy of the E3 fragment to obtain PAZ1.

[0041] DENe3: Fragment of the envelope protein of DEN-3.

[0042] N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.

[0043]FIG. 8. Cloning strategy of the E3 fragment to obtain PAZ2.

[0044] DENe3: Fragment of the envelope protein of DEN-3.

[0045] MDH: dehydrogenase mutant.

[0046]FIG. 9. Cloning strategy of the E3 fragment to obtain PAZ3.

[0047] DENe3: Fragment of the envelope protein of DEN-3.

[0048] MDH: dehydrogenase mutant.

[0049]FIG. 10. Cloning strategy of the E4 fragment to obtain PID1.

[0050] DENe4: Fragment of the envelope protein of DEN-4.

[0051] N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.

[0052]FIG. 11. Cloning strategy of the E4 fragment to obtain PID2.

[0053] DENe4: Fragment of the envelope protein of DEN4.

[0054] MDH: dehydrogenase mutant.

[0055]FIG. 12. Cloning strategy of the E4 fragment to obtain PID3.

[0056] DENe4: Fragment of the envelope protein of DEN4.

[0057] MDH: dehydrogenase mutant.

[0058]FIG. 13. Cloning strategy to obtain PD4D2.

[0059] DENe4: Fragment of the envelope protein of DEN4.

[0060] DENe2: Fragment of the envelope protein of DEN-2.

[0061] MDH: dehydrogenase mutant.

EXAMPLES OF PERFORMANCE Example 1 Obtaining of PLL1

[0062] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-2 virus (Sec. Id. No. 22) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.1 and Sequence No. 2 from the DEN-2 virus strain genotype Jamaica (Deubel V., Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Jamaica genotype: Comparative analysis of the full-length genome. Virology 1988.165:234-244).

[0063] The vector was created by digestion of the pM108 His plasmid with Xba I/Bam HI, which contains the nucleotide sequence that codifies for the N-terminal region of the MDH and for a sequence of 6 histidines (Sequence No. 23). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 (Hill C. W., Harnish B. W. 1982. Transposition of a chromosomal segment bounded by redundant rRNA genes in Escherichia coli. J Bacteriology. 149:449-457) were transformed with the selected clone, called pLL1 (FIG. 1 and Sequence No. 24). Upon growing the colony in Luria Bertani (LB) medium, a SDS-PAGE of the cellular lysate was done. As a result a 25 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the N-terminal region from the MDH protein and the DENe protein fragment from the DEN-2 virus. The protein was recognized in Immunoblotting by polyclonal antibodies (PA) anti-DEN-2 contained in the HMAF. This protein was denominated PLL1 (Sequence No. 25).

Example 2 Purification of the PLL1 Protein

[0064] The biomass obtained from the E. coli strain transformed with pLL1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained preponderantly as insoluble form associated to the pellet of the cellular disruption. From the pellet the protein was extracted with urea 6 M and the supernatant, containing the PLL1 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The protein was eluted with Imidazole 50 mM and the obtained volume was loaded onto a G-25 column to finally obtain the protein in the formulation buffer NaCl 100 mM, KCl₂ 2 mM, Na₂HPO₄ 10 mM, pH 7.2, KH₂PO₄ 1 mM (PBS). This preparation was used for immunological studies.

Example 3 Antigenic characterization of PLL1

[0065] The purified fraction of PLL1 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 1). TABLE 1 Reactivity of PLL1 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PLL1* HMAF DEN-1 + HMAF DEN-2 ++ HMAF DEN-3 − HMAF DEN-4 − HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT +

[0066] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-2. This result coincides with the fact that the cloned region belongs to serotype 2. The recognition by HMAF against the other serotypes was less than in the case of serotype 2, in decreasing order: DEN-1, DEN-3 and DEN4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. On the other hand, the Mab 3H5 had reactivity indeed. This recognition by Western blotting relied on disulphide bond since when the sample was reduced the signal was lost. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-2 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting.

Example 4 Characterization of the Antibody Response Generated by PLL1

[0067] A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PLL1 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-2 were obtained while, no reactivity was obtained against the rest of the serotypes (table 2 and table 5). In addition, the hemagglutination inhibition assay (HIA) was done and only positive titers were found against DEN-2 (table 3 and table 5). Finally, the in vitro neutralization assay was done and neutralization titers of 1:320 against DEN-2 were obtained. However, no neutralization of the viral infection was found against the rest of the serotypes (table 4 and table 5). These results indicate the high serotype-specificity of the antibodies elicited by PLL1. TABLE 2 Antibody titers against DEN-2 from the sera obtained upon immunization of mice with PLL1. Titers Titers anti-DEN-2 anti-DEN-2 Mouse PLL1 PBSControl (−) 1 1/128 000 <1:100 2 1/64 000 <1:100 3 1/64 000 <1:100 4 1/128 000 <1:100 5 1/32 000 <1:100 6 1/32 000 <1:100 7 1/64 000 <1:100 8 1/32 000 <1:100 9 1/128 000 <1:100 10 1/512 000 <1:100

[0068] TABLE 3 Titers by HI of the sera from the animals immunized with PLL1. Titers by Titers by HI* anti-DEN-2 HI anti-DEN-2 Mouse PLL1 PBS C(−) 1 <1:5 <1:5 2 >1:640 <1:5 3  1:320 <1:5 4  1:320 <1:5 5 >1:640 <1:5 6 >1:640 <1:5 7 >1:640 <1:5 8  1:320 <1:5 9 >1:640 <1:5 10 <1:5 <1:5

[0069] TABLE 4 Viral neutralization assay with the sera of animals immunized with PLL1. Neutralizing Neutralizing titers* titers anti-DEN-2 anti-DEN-2 Mouse PLL1 PBS C(−) 1 1:320 <1:5 2 1:320 <1:5 3 1:320 <1:5 4 1:320 <1:5 5 1:80 <1:5 6 1:160 <1:5 7 1:320 <1:5 8 1:40 <1:5 9 1:160 <1:5 10 1:320 <1:5

[0070] TABLE 5 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PLL1. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1 (PLL1) <1/100 >1:128 000 <1/100 <1/100 2 (PLL1) <1/100  1:128 000 <1/100 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PLL1 <1/5 >1/320 <1/5 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1 (PLL1) <1:5 1:320 <1:5 <1:5 2 (PLL1) <1:5 1:160 <1:5 <1:5

Example 5 Obtaining of PLL2

[0071] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-2 virus (Sec. Id. No. 22) was amplified with the oligonucleotides identified in the list of sequences as Sequence No. 1 and Sequence No. 3 from the DEN-2 virus strain genotype Jamaica (Deubel V., Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Jamaica genotype: Comparative analysis of the full-length genome. Virology 1988.165:234-244).

[0072] The vector was created by digestion of the pM84 His plasmid with Xba I/Eco RI, which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines (Sequence No. 26). This digestion permits the insertion of the amplified fragment by PCR within the coding region for a structural domain of the MDH protein.

[0073] Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 (Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580) were transformed with the selected clone, called pLL2 (FIG. 2 and Sequence No. 27). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-2 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-2 and was denominated PLL2 (Sequence No. 28).

Example 6 Purification of the PLL2 Protein

[0074] The biomass obtained from the E. coli strain transformed with pLL2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the soluble fraction a metal ions-affinity chromatography was done using the Chelating-sepharose FF column previously coupled with Cu⁺⁺ ions. The column was washed using Imidazolee 15 mM and the protein was eluted with Imidazolee 100 mM. On the other hand, the protein associated to the insoluble fraction was extracted using Urea 8 M, and the supernatant, containing the PLL2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The protein was eluted with Imidazolee 100 mM. Finally, the pure fraction of each form of the protein were loaded onto a G-25 column to obtain the protein in the formulation buffer NaCl 100 mM, KCl₂ 2 mM, Na₂HPO₄ 10 mM, pH 7.2, KH₂PO₄ 1 mM (PBS). This preparations were used for immunological studies.

Example 7 Antigenic characterization of PLL2

[0075] The purified fraction of PLL2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 6).

[0076] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-2. The recognition by HMAF against the other serotypes was less than in the case of serotype 2, in decreasing order: DEN-1, DEN-3 and DEN4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Nevertheless, related to the Mab 3H5, a great reactivity was observed (even higher than those obtained with the PLL1) either by Dot blot and Western blot. Contrariously to the PLL1 results, the recognition with the Mab 3H5 was the same when the reducing agents are present in the sample, indicating a possible conformational difference between both proteins. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-2 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting. TABLE 6 Reactivity of PLL2 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PLL2 sol, ins* HMAF DEN-1 ++ HMAF DEN-2 +++ HMAF DEN-3 − HMAF DEN-4 − HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT +++

Example 8 Characterization of the Antibody Response Generated by PLL2

[0077] A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PLL2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-2 were obtained while, no reactivity was obtained against the rest of the serotypes (table 7 and table 10). In addition, the hemagglutination inhibition assay (HI) was done and only positive titers were found against DEN-2 (table 8 and table 10). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-2 were obtained. Similarly to the results obtained with PLL1, no neutralization of the viral infection was found against the rest of the serotypes (table 9 and table 10). On the other hand, the results obtained with both variants of PLL2 were similar, indicating that the solubility status of the protein do not influece in the capacity of generating functional antibodies. TABLE 7 Antibody titers against DEN-2 from the sera obtained upon immunization of mice with PLL2 soluble and insoluble. Titers Titers anti-DEN-2 (PLL2) anti-DEN-2 Mouse PLL2 s PLL2 ins PBS C(−) 1 >1:128 000  1:64000 <1:100 2  1:128 000 >1:128 000 <1:100 3 >1:128 000  1:128 000 <1:100 4 >1:128 000 >1:128 000 <1:100 5  1:64 000 >1:128 000 <1:100 6 >1:128 000  1:128 000 <1:100 7  1:64000 >1:128 000 <1:100 8 >1:128 000  1:64000 <1:100 9 >1:128 000  1:64000 <1:100 10  1:128 000 >1:128 000 <1:100

[0078] TABLE 8 Titers by HI of the sera from the animals immunized with PLL2 soluble and insoluble. Titers by HI* Titers by HI anti-DEN-2 (PLL2) anti-DEN-2 Mouse PLL2 s PLL2 ins PBS C(−) 1 >1:640 >1:640 <1:5 2 >1:640 >1:640 <1:5 3  1:320 >1:640 <1:5 4 >1:640  1:320 <1:5 5  1:320 <1:5 <1:5 6 >1:640 >1:640 <1:5 7 >1:640  1:320 <1:5 8 <1:5  1:320 <1:5 9  1:320 >1:640 <1:5 10 >1:640 >1:640 <1:5

[0079] TABLE 9 Viral neutralization assay with the sera of animals immunized with PLL2 soluble and insoluble. Neutralizing Neutralizing titers* titers anti-DEN-2 (PLL2) anti-DEN-2 Mouse PLL2 s PLL2 ins PBS C(−) 1 >1:1280 >1:1280 >1:1280 2 >1:1280 >1:1280 <1:5 3 >1:1280  1:640 <1:5 4  1:640 >1:1280 <1:5 5  1:640  1:640 <1:5 6 >1:1280 >1:1280 <1:5 7 >1:1280 >1:1280 <1:5 8 >1:1280 >1:1280 <1:5 9 >1:1280 >1:1280 <1:5 10 >1:1280 >1:1280 <1:5

[0080] TABLE 10 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PLL2 soluble and insoluble. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1 (PLL2 sol.) <1/100 >1:128 000 <1/100 <1/100 2 (PLL2 sol.) <1/100  1:64000 <1/100 <1/100 1 (PLL2 ins.) <1/100  1:64000 <1/100 <1/100 2 (PLL2 ins.) <1/100 >1:128 000 <1/100 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PLL2 sol. <1/5 >1/320 <1/5 <1/5 PLL2 ins. <1/5 >1/320 <1/5 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1 (PLL2) <1:5 1:320 <1:5 <1:5 2 (PLL2) <1:5 1:160 <1:5 <1:5

Example 9 Obtaining of pLL3

[0081] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-2 virus (Sec. Id. No. 22) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.4 and Sequence No.5 from the DEN-2 virus strain genotype Jamaica (Deubel V., Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Jamaica genotype: Comparative analysis of the full-length genome. Virology 1988.165:234-244).

[0082] The vector was created by digestion of the pD4 plasmid with Bam HI/Bam HI which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines without stop codon (Sequence No. 29). This digestion permits to the fusion of the amplified fragment by PCR after the C-terminal region for the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pLL3 (FIG. 3 and Sequence No. 30). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-2 virus. The protein was recognized in Immunoblotting by a HMAFI anti-DEN-2 and was denominated PLL3 (Sequence No. 31).

Example 10 Purification of the PLL3 Protein

[0083] The biomass obtained from the E. coli strain transformed with pLL2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the insoluble fraction, the protein was extracted using Urea 6 M, and the supernatant, containing the PLL3 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-Sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The colum was washed with Imidazolee 30 mM and the protein was eluted with Imidazole 100 mM. Finally, the pure fraction of the protein was loaded onto a G-25 column to obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.

Example 11 Antigenic Characterization of PLL3

[0084] The purified fraction of PLL3 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 11).

[0085] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-2. The recognition by HMAF against the other serotypes was lower than in the case of serotype 2, in decreasing order: DEN-1, DEN-3 and DEN-4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Nevertheless, related to the Mab 3H5, a great reactivity was observed (similar to those obtained with the PLL2) either by Dot blot and Western blot. Contrariously to the PLL1 results, the recognition with the Mab 3H5 was the same when the reducing agents are present in the sample, indicating a possible conformational difference between both proteins. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-2 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting. These results were similar to those obtained with PLL2. TABLE 11 Reactivity of PLL3 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PLL3* HMAF DEN-1 ++ HMAF DEN-2 +++ HMAF DEN-3 − HMAF DEN-4 − HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT +++

Example 12 Characterization of the Antibody Response Generated by PLL3

[0086] A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PLL3 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-2 were obtained while, no reactivity was obtained against the rest of the serotypes (table 12 and table 15). In addition, the hemagglutination inhibition (HI) assay was done and only positive titers were found against DEN-2 (table 13 and table 15). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-2 were obtained (table 14). No neutralization of the viral infection was found against the rest of the serotypes (table 15). Using the three tests, high levels of serotype-specific antibodies were detected, similar of those obtained after the immunization with the PLL2 protein. TABLE 12 Antibody titers against DEN-2 from the sera obtained upon immunization of mice with PLL3. Titers Titers anti-DEN-2 anti-DEN-2 Mouse (PLL3) PBS C(−) 1  1:128 000 <1:100 2 >1:128 000 <1:100 3 >1:128 000 <1:100 4  1:64 000 <1:100 5 >1:128 000 <1:100 6  1:64 000 <1:100 7 >1:128 000 <1:100 8 >1:128 000 <1:100 9  1:128 000 <1:100 10 >1:128 000 <1:100

[0087] TABLE 13 Titers by HI of the sera from the animals immunized with PLL3. Titers by HI* Titers by HI anti-DEN-2 anti-DEN-2 Mouse (PLL3) PBS C(−) 1 >1:640 <1:5 2  1:320 <1:5 3  1:320 <1:5 4 >1:640 <1:5 5  1:320 <1:5 6 >1:640 <1:5 7 >1:640 <1:5 8 >1:640 <1:5 9  1:320 <1:5 10 >1:640 <1:5

[0088] TABLE 14 Viral neutralization assay with the sera of animals immunized with PLL3. Neutralizing Neutralizing titers titers anti-DEN-2 anti-DEN-2 Mouse PLL3 PBS C(−) 1 >1:1280 <1:5 2 >1:1280 <1:5 3 >1:1280 <1:5 4  1:640 <1:5 5 >1:1280 <1:5 6 >1:1280 <1:5 7 >1:1280 <1:5 8 >1:1280 <1:5 9  1:640 <1:5 10 >1:1280 <1:5

[0089] TABLE 15 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PLL3. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1(PLL3) <1/100 >1:128000 <1/100 <1/100 2(PLL3) <1/100 >1:128000 <1/100 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PLL3 <1/5 >1/320 <1/5 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers*** titers*** titers*** of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PLL3) <1:5 >1:1280 <1:5 <1:5 2(PLL3) <1:5 >1:1280 <1:5 <1:5

Example 13 Obtaining of pLH1

[0090] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-1 virus (Sec. Id. No. 32) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.6 and Sequence No.7 from the DEN-1 virus strain genotype (Chu M. C., O'Rourke E. J., Trent D. W. Genetic relatedness among structural protein genes of dengue 1 virus strains. J. Gen. Virol. 1989. 70:1701-1712).

[0091] The vector was created by digestion of the pM108 His plasmid with Xba I/Bam HI, which contains the nucleotide sequence that codifies for the N-terminal region of the MDH and for a sequence of 6 histidines (Sequence No. 23). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pLH1 (FIG. 4 and Sequence No.33). Upon growing the colony in Luria Bertani (LB) medium, a SDS-PAGE of the cellular lysate was done. As a result a 25 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the N-terminal region from the MDH protein and the DENe protein fragment from the DEN-1 virus. The protein was recognized in Immunoblotting by polyclonal antibodies (PA) anti-DEN-1 contained in the HMAF. This protein was denominated PLH1 (Sequence No.34).

Example 14 Purification of the Protein PLH1

[0092] The biomass obtained from the E. coli strain transformed with pLH1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained preponderantly as insoluble form associated to the pellet of the cellular disruption. From the pellet, the protein was extracted with urea 7 M and the supernatant, containing the PLH1 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The protein was eluted with Imidazolee 60 mM and the obtained volume was loaded onto a G-25 column to finally obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.

Example 15 Antigenic Characterization of PLH1

[0093] The purified fraction of PLH1 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 16).

[0094] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-1. The recognition by HMAF against the other serotypes was lower than in the case of serotype 1. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-1 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting. TABLE 16 Reactivity of PLH1 protein to monoclonal and polyclonal antibodies Abs** Specificity*** PLH1 HMAF DEN-1 ++ HMAF DEN-2 + HMAF DEN-3 − HMAF DEN-4 − HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 16 Characterization of the Antibody Response Generated by PLH1

[0095] A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PLH1 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 17 and table 20). In addition, the HI assay was done and only positive titers were found against DEN-1 (table 18 and table 20). Finally, the in vitro neutralization assay was done and neutralization titers of 1:320 against DEN-1 were obtained. However, no neutralization of the viral infection was found against the rest of the serotypes (table 19 and table 20). These results indicate the high serotype-specificity of the antibodies elicited by PLH1. TABLE 17 Antibody titers against DEN-1 from the sera obtained upon immunization of mice with PLH1. Titers Titers anti-DEN-1 anti-DEN-1 Mouse (PLH1) PBS C(−) 1 1/64 000 <1:100 2 1/128 000 <1:100 3 1/64 000 <1:100 4 1/32 000 <1:100 5 1/32 000 <1:100 6 1/64 000 <1:100 7 1/128 000 <1:100 8 1/64 000 <1:100 9 1/128 000 <1:100 10 1/128 000 <1:100

[0096] TABLE 18 Titers by HI of the sera from the animals immunized with PLH1 Titers by HI* Titers by HI* anti-DEN-1 anti-DEN-1 Mouse (PLH1) PBS C(−) 1 >1:640 <1:5 2  1:320 <1:5 3 >1:640 <1:5 4 >1:640 <1:5 5 >1:640 <1:5 6  1:320 <1:5 7  1:40 <1:5 8  1:320 <1:5 9 >1:640 <1:5 10 <1:5 <1:5

[0097] TABLE 19 Viral neutralization assay with the sera of animals immunized with PLH1. Neutralizing Neutralizing titers* titers* anti-DEN-1 anti-DEN-1 Mouse (PLH1) PBS C(−) 1 1:80 <1:5 2 1:320 <1:5 3 1:40 <1:5 4 1:320 <1:5 5 1:80 <1:5 6 1:160 <1:5 7 1:320 <1:5 8 1:320 <1:5 9 1:320 <1:5 10 1:320 <1:5

[0098] TABLE 20 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PLH1. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1 (PLH1)  1:128 000 <1/100 <1/100 <1/100 2 (PLH1) >1:128 000 <1/100 <1/100 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PLH1 >1:320 <1/5 <1/5 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers*** titers*** titers*** of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PLH1) 1:160 <1:5 <1:5 <1:5 2(PLH1) 1:320 <1:5 <1:5 <1:5

Example 17 Obtaining of pLH2

[0099] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-1 virus (Sequence No. 32) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.6 and Sequence No.8 from a DEN-1 viral strain (Chu M. C., O'Rourke E. J., Trent D. W. Genetic relatedness among structural protein genes of dengue 1 virus strains. J. Gen. Virol. 1989. 70:1701-1712).

[0100] The vector was created by digestion of the pM84 His plasmid with Xba I/Eco RI, which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines (Sequence No. 26). This digestion permits the insertion of the amplified fragment by PCR within the coding region for a structural domain of the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 were transformed with the selected clone, called pLH2 (FIG. 5 and Sequence No.35). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-1 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-1 and was denominated PLH2 (Sequence No.36).

Example 18 Purification of the PLH2 Protein

[0101] The biomass obtained from the E. coli strain transformed with pLH2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. The protein associated to the insoluble fraction was extracted using Urea 7 M, and the supernatant, containing the PLH2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The column was washed with Imidazolee 40 mM and the protein was eluted with Imidazole 100 mM. Finally, the pure fraction was loaded onto a G-25 column to obtain the protein in the formulation (PBS). This preparation was used for immunological studies.

Example 19 Antigenic Characterization of PLH2

[0102] The purified fraction of PLH2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 21).

[0103] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-1 (higher than those obtained with PLH1). The recognition by HMAF against the other serotypes was lower than in the case of serotype 1. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against five human sera of high titers and three of low titers against DEN-1 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting. TABLE 21 Reactivity of PLH2 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PLH2 HMAF DEN-1 +++ HMAF DEN-2 + HMAF DEN-3 − HMAF DEN-4 − HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 20 Characterization of the Antibody Response Generated by PLH2

[0104] A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PLH2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 22 and table 25). In addition, the HI assay was done and only positive titers were found against DEN-1 (table 23 and table 25). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-1 were obtained (table 24). No neutralization of the viral infection was found against the rest of the serotypes. (table 25). TABLE 22 Antibody titers against DEN-1 from the sera obtained upon immunization of mice with PLH2. Titers Titers anti-DEN-1 anti-DEN-1 Mouse (PLH2) PBS C(−) 1  1:128 000 <1:100 2 >1:128 000 <1:100 3  1:64000 <1:100 4 >1:128 000 <1:100 5  1:128 000 <1:100 6  1:64000 <1:100 7 >1:128 000 <1:100 8  1:128 000 <1:100 9 >1:128 000 <1:100 10 >1:128 000 <1:100

[0105] TABLE 23 Titers by HI of the sera from the animals immunized with PLH2. Titers by HI* Titers by HI anti-DEN-1 anti-DEN-1 Mouse (PLH2) PBS C(−) 1 <1:5 <1:5 2 >1:640 <1:5 3  1:320 <1:5 4  1:320 <1:5 5 >1:640 <1:5 6 >1:640 <1:5 7 >1:640 <1:5 8  1:320 <1:5 9 >1:640 <1:5 10 <1:5 <1:5

[0106] TABLE 24 Viral neutralization assay with the sera of animals immunized with PLH2. Neutralizing Neutralizing titers* titers* anti-DEN-1 anti-DEN-1. Mouse (PLH2) C(−) 1 >1:1280 <1:5 2 >1:1280 <1:5 3 >1:1280 <1:5 4 >1:1280 <1:5 5 >1:1280 <1:5 6 >1:1280 <1:5 7 >1:1280 <1:5 8 >1:1280 <1:5 9  1:640 <1:5 10 >1:1280 <1:5

[0107] TABLE 25 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PLH2. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1 (PLH2)  1:64 000 <1/100 <1/100 <1/100 2 (PLH2) >1:128 000 <1/100 <1/100 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PLH2 >1/320 <1/5 <1/5 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PLH2) >1:1280 <1:5 <1:5 <1:5 2(PLH2) >1:1280 <1:5 <1:5 <1:5

Example 21 Obtaining of pLH3

[0108] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-1 virus (Sec. Id. No. 32) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.9 and Sequence No.10 from the DEN-1 virus strain (Chu M. C., O'Rourke E. J., Trent D. W. Genetic relatedness among structural protein genes of dengue 1 virus strain. J. Gen. Virol.1989. 70:1701-1712).

[0109] The vector was created by digestion of the pD4 plasmid with Bam HI/Bam HI which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines without stop codon (Sequence No.29). This digestion permits the fusion of the amplified fragment by PCR after the C-terminal region for the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pLH3 (FIG. 6 and Sequence No.37). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-1 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-1 and was denominated PLH3 (Sequence No.38).

Example 22 Purification of the PLH3 Protein

[0110] The biomass obtained from the E. coli strain transformed with pLH3 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the insoluble fraction, the protein was extracted using Urea 6 M, and the supernatant, containing the PLH3 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-Sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The colum was washed with Imidazolee 30 mM and the protein was eluted with Imidazole 250 mM. Finally, the pure fraction of the protein was loaded onto a G-25 column to obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.

Example 23 Antigenic characterization of PLH3

[0111] The purified fraction of PLH3 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 26).

[0112] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-1. The recognition by HMAF against the other serotypes was lower than in the case of serotype 1. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-1 was measured, achieving a substantial signal in both cases by Dot blotting and Western blotting. TABLE 26 Reactivity of PLH3 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PLH3 HMAF DEN-1 +++ HMAF DEN-2 + HMAF DEN-3 − HMAF DEN-4 − HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 24 Characterization of the Antibody Response Generated by PLH3

[0113] A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PLH3 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 27 and table 30). In addition, the HI assay was done and only positive titers were found against DEN-1 (table 28 and table 30). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-1 were obtained (table 29). No neutralization of the viral infection was found against the rest of the serotypes (table 30). TABLE 27 Antibody titers against DEN-1 from the sera obtained upon immunization of mice with PLH3. Titers Titers anti-DEN-1 anti-DEN-1 Mouse PLH3 PBSControl (−) 1  1:64 000 <1:100 2 >1:128 000 <1:100 3  1:64 000 <1:100 4 >1:128 000 <1:100 5  1:128 000 <1:100 6  1:128 000 <1:100 7  1:128 000 <1:100 8 >1:128 000 <1:100 9  1:64 000 <1:100 10 >1:128 000 <1:100

[0114] TABLE 28 Titers by HI of the sera from the animals immunized with PLH3. Titers by HI* Titers by HI anti-DEN-1 anti-DEN-1 Mouse PLH3 PBS C(−) 1  1:320 <1:5 2 >1:640 <1:5 3 >1:640 <1:5 4  1:320 <1:5 5  1:320 <1:5 6 >1:640 <1:5 7  1:320 <1:5 8 <1:5 <1:5 9 >1:640 <1:5 10 >1:640 <1:5

[0115] TABLE 29 Viral neutralization assay with the sera of animals immunized with PLH3. Neutralizing Neutralizing titers* titers* anti-DEN-1 anti-DEN-1 Mouse PLH3 PBS C(−) 1 >1:1280 <1:5 2 >1:1280 <1:5 3 >1:1280 <1:5 4  1:640 <1:5 5  1:640 <1:5 6 >1:1280 <1:5 7 >1:1280 <1:5 8 >1:1280 <1:5 9 >1:1280 <1:5 10 >1:1280 <1:5

[0116] TABLE 30 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PLH3. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) anti DEN-2) anti-DEN-3) (anti-DEN- 4) 1(PLH3)  1:128 000 <1/100 <1/100 <1/100 2(PLH3) >1:128 000 <1/100 <1/100 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PLH3 >1/320 <1/5 <1/5 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PLH3) >1:1280 <1:5 <1:5 <1:5 2(PLH3) >1:1280 <1:5 <1:5 <1:5

Example 25 Obtaining of pAZ1

[0117] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-3 virus (Seq. 39) was amplified with the oligonucleotides identified in the list of sequences as Sequence No. 11 and Sequence No.12 from the DEN-3 virus strain genotype (Osatomi K., Sumiyoshi H. Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology.1990. 176(2):643-647).

[0118] The vector was created by digestion of the pM108 His plasmid with Xba I/Bam HI, which contains the nucleotide sequence that codifies for the N-terminal region of the MDH and for a sequence of 6 histidines (Sequence No. 23). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pAZ1 (FIG. 7 and Sequence No.40). Upon growing the colony in Luria Bertani (LB) medium, a SDS-PAGE of the cellular lysate was done. As a result a 25 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the N-terminal region from the MDH protein and the DENe protein fragment from the DEN-3 virus. The protein was recognized in Immunoblotting by polyclonal antibodies (PA) anti-DEN-3 contained in the HMAF. This protein was denominated PAZ1 (Sequence No.41).

Example 26 Purification of the Protein PAZ1

[0119] The biomass obtained from the E. coli strain transformed with pAZ1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained preponderantly as insoluble form associated to the pellet of the cellular disruption. From the pellet, the protein was extracted with urea 7 M and the supernatant, containing the PLH1 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The protein was eluted with Imidazolee 60 mM and the obtained volume was loaded onto a G-25 column to finally obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.

Example 27 Antigenic Characterization of PAZ1

[0120] The purified fraction of PAZ1 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 31).

[0121] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-3. The recognition by HMAF against the other serotypes was lower than in the case of serotype 3. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-3 was measured, achieving a substantial signal in both cases by Dot blotting Western blotting. TABLE 31 Reactivity of PAZ1 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PAZ1 HMAF DEN-1 − HMAF DEN-2 + HMAF DEN-3 ++ HMAF DEN-4 + HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 28 Characterization of the Antibody Response Generated by PAZ1

[0122] A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PAZ1 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 32 and table 35). In addition, the HI assay was done and only positive titers were found against DEN-3 (table 33 and table 35). Finally, the in vitro neutralization assay was done and neutralization titers of 1:320 against DEN-3 were obtained. However, no neutralization of the viral infection was found against the rest of the serotypes (table 34 and table 35). These results indicate the high serotype-specificity of the antibodies elicited by PAZ1. TABLE 32 Antibody titers against DEN-3 from the sera obtained upon immunization of mice with PAZ1. Titers Titers anti-DEN-3 anti-DEN-3 Mouse PAZ1 PBSControl (−) 1 1/64 000 <1:100 2 1/128 000 <1:100 3 1/32 000 <1:100 4 1/64 000 <1:100 5 1/64 000 <1:100 6 1/128 000 <1:100 7 1/64 000 <1:100 8 1/64 000 <1:100 9 1/128 000 <1:100 10 1/128 000 <1:100

[0123] TABLE 33 Titers by HI of the sera from the animals immunized with PAZ1. Titers by HI* Titers by HI anti-DEN-3 anti-DEN-3 Mouse PAZ1 PBS C(−) 1 >1:640 <1:5 2  1:320 <1:5 3  1:320 <1:5 4  1:640 <1:5 5 <1/5 <1:5 6  1:320 <1:5 7 <1/5 <1:5 8  1:320 <1:5 9 >1:640 <1:5 10 >1:640 <1:5

[0124] TABLE 34 Viral neutralization assay with the sera of animals immunized with PAZ1. Neutralizing Neutralizing titers titers anti-DEN-3 anti-DEN-3 Mouse PAZ1 PBS C(−) 1 1:160 <1:5 2 1:320 <1:5 3 1:320 <1:5 4 1:320 <1:5 5 1:40 <1:5 6 1:40 <1:5 7 1:320 <1:5 8 1:320 <1:5 9 1:160 <1:5 10 1:320 <1:5

[0125] TABLE 35 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PAZ1. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti-DEN-2) (anti-DEN-3) (anti-DEN-4) 1 (PAZ1) <1/100 <1/100  1:64 000 <1/100 2 (PAZ1) <1/100 <1/100 >1:128000 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PAZ1 <1/5 <1/5 >1:320 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PAZ1) <1:5 <1:5 1:320 <1:5 2(PAZ1) <1:5 <1:5 1:320 <1:5

Example 29 Obtaining of pAZ2

[0126] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-3 virus (Sequence No.39) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.11 and Sequence No.13 from a DEN-3 viral strain (Osatomi K., Sumiyoshi H. Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology. 1990.176(2):643-647).

[0127] The vector was created by digestion of the pM84 His plasmid with Xba I/Eco RI, which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines (Sequence No. 26). This digestion permits the insertion of the amplified fragment by PCR within the coding region for a structural domain of the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 were transformed with the selected clone, called pAZ2 (FIG. 8 and Sequence No.42). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-3 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-3 and was denominated PAZ2 (Sequence No.43).

Example 30 Purification of the PAZ2 Protein

[0128] The biomass obtained from the E. coli strain transformed with pAZ2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. The protein associated to the insoluble fraction was extracted using Urea 7 M, and the supernatant, containing the PAZ2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The column was washed with Imidazolee 40 mM and the protein was eluted with Imidazole 100 mM. Finally, the pure fraction was loaded onto a G-25 column to obtain the protein in the formulation (PBS). This preparation was used for immunological studies.

Example 31 Antigenic Characterization of PAZ2

[0129] The purified fraction of PAZ2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 36).

[0130] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-3 (higher than those obtained with PAZ1). The recognition by HMAF against the other serotypes was lower than in the case of serotype 3. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against five human sera of high titers and three of low titers against DEN-3 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting. TABLE 36 Reactivity of PAZ2 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PAZ2 HMAF DEN-1 − HMAF DEN-2 − HMAF DEN-3 +++ HMAF DEN-4 + HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 32 Characterization of the Antibody Response Generated by PAZ2

[0131] A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PAZ2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-3 were obtained while, no reactivity was obtained against the rest of the serotypes (table 37 and table 40). In addition, the HI assay was done and only positive titers were found against DEN-3 (table 38 and table 40). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-3 were obtained (table 39). No neutralization of the viral infection was found against the rest of the serotypes (table 40). TABLE 37 Antibody titers against DEN-3 from the sera obtained upon immunization of mice with PAZ2. Titers Titers anti-DEN-3 anti-DEN-3 Mouse PAZ2 PBS Control (−) 1 >1:128 000 <1:100 2  1:128 000 <1:100 3 >1:128 000 <1:100 4 >1:128 000 <1:100 5  1:128 000 <1:100 6 >1:128 000 <1:100 7  1:64000 <1:100 8 >1:128 000 <1:100 9  1:64000 <1:100 10 >1:128 000 <1:100

[0132] TABLE 38 Titers by HI of the sera from the animals immunized with PAZ2. Titers Titers anti-DEN-3 anti-DEN-3 Mouse PAZ2 PBS 1 >1:640 <1:5 2  1:320 <1:5 3 >1:640 <1:5 4 >1:640 <1:5 5 >1:640 <1:5 6  1:320 <1:5 7 <1:5 <1:5 8  1:320 <1:5 9 >1:640 <1:5 10 >1:640 <1:5

[0133] TABLE 39 Viral neutralization assay with the sera of animals immunized with PAZ2. Neutralizing Neutralizing titers titers anti-DEN-3 anti-DEN-3 Mouse PAZ2 PBS C(−) 1 >1:1280 <1:5 2 >1:1280 <1:5 3 >1:1280 <1:5 4 >1:1280 <1:5 5 >1:1280 <1:5 6  1:640 <1:5 7 >1:1280 <1:5 8  1:640 <1:5 9 >1:1280 <1:5 10  1:640 <1:5

[0134] TABLE 40 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PAZ2. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1(PAZ2) <1/100 <1/100 >1:128 000 <1/100 2(PAZ2) <1/100 <1/100 >1:128 000 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PAZ2 <1/5 <1/5 >1/320 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PAZ2) <1:5 <1:5 >1:1280 <1:5 2(PAZ2) <1:5 <1:5 >1:1280 <1:5

Example 33 Obtaining of pAZ3

[0135] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-3 virus (Seq. 39) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.14 and Sequence No.15 from the DEN-3 viral strain (Osatomi K., Sumiyoshi H. Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology.1990. 176(2):643-647).

[0136] The vector was created by digestion of the pD4 plasmid with Bam HI/Bam HI which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines without stop codon (Sequence No.29). This digestion permits the fusion of the amplified fragment by PCR after the C-terminal region for the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pAZ3 (FIG. 9 and Sequence No.44). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-3 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-3 and was denominated PAZ3 (Sequence No.45).

Example 34 Purification of the PAZ3 Protein

[0137] The biomass obtained from the E. coli strain transformed with pAZ3 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the insoluble fraction, the protein was extracted using Urea 7 M, and the supernatant, containing the PAZ3 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-Sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The colum was washed with Imidazolee 45 mM and the protein was eluted with Imidazole 230 mM. Finally, the pure fraction of the protein was loaded onto a G-25 column to obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.

Example 35 Antigenic Characterization of PAZ3

[0138] The purified fraction of PAZ3 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 26).

[0139] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-3. The recognition by HMAF against the other serotypes was lower than in the case of serotype 3. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-3 was measured, achieving a substantial signal in both cases by Dot blotting and Western blotting. TABLE 41 Reactivity of PAZ3 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PAZ3 HMAF DEN-1 − HMAF DEN-2 − HMAF DEN-3 +++ HMAF DEN-4 + HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 36 Characterization of the Antibody Response Generated by PAZ3

[0140] A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PAZ3 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-3 were obtained while, no reactivity was obtained against the rest of the serotypes (table 42 and table 45). In addition, the HI assay was done and only positive titers were found against DEN-3 (table 43 and table 45). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-3 were obtained (table 44). No neutralization of the viral infection was found against the rest of the serotypes (table 45). TABLE 42 Antibody titers against DEN-3 from the sera obtained upon immunization of mice with PAZ3. Titers Titers anti-DEN-3 anti-DEN-3 Mouse PAZ3 PBSControl (−) 1 >1:128 000 <1:100 2  1:128 000 <1:100 3 >1:128 000 <1:100 4  1:128000 <1:100 5  1:128 000 <1:100 6 >1:128 000 <1:100 7 >1:128 000 <1:100 8  1:128 000 <1:100 9  1:128 000 <1:100 10 >1:128 000 <1:100

[0141] TABLE 43 Titers by HI of the sera from the animals immunized with PAZ3. Titers by HI* Titers by HI anti-DEN-3 anti-DEN-3 Mouse PAZ3 PBS C(−) 1 >1:640 <1:5 2 >1:640 <1:5 3  1:320 <1:5 4 <1:5 <1:5 5 >1:640 <1:5 6 <1:5 <1:5 7  1:320 <1:5 8 >1:640 <1:5 9 >1:640 <1:5 10  1:320 <1:5

[0142] TABLE 44 Viral neutralization assay with the sera of animals immunized with PAZ3. Neutralizing Neutralizing titers titers anti-DEN-3 anti-DEN-3 Mouse PAZ3 PBS C(−) 1 >1:1280 <1:5 2  1:640 <1:5 3 >1:1280 <1:5 4 >1:1280 <1:5 5 >1:1280 <1:5 6 >1:1280 <1:5 7 >1:1280 <1:5 8 >1:1280 <1:5 9 >1:1280 <1:5 10 >1:1280 <1:5

[0143] TABLE 45 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PAZ3. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1(PAZ3) <1/100 <1/100 >1:128 000 <1/100 2(PAZ3) <1/100 <1/100  1:128 000 <1/100 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PAZ3 <1/5 <1/5 >1/320 <1/5 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PAZ3) <1:5 <1:5 >1:1280 <1:5 2(PAZ3) <1:5 <1:5 >1:1280 <1:5

Example 37 Obtaining of pID1

[0144] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-4 virus (Sequence No.46) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.17 and Sequence No.18 from the DEN4 virus strain genotype (Zhao B., Mackow E. R., Buckler-White A. J., Markoff L., Chancock R. M., Lai C. -J., Makino Y. Cloning full-length Dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 1986. 155:77-88).

[0145] The vector was created by digestion of the pM108 His plasmid with Xba I/Bam HI, which contains the nucleotide sequence that codifies for the N-terminal region of the MDH and for a sequence of 6 histidines (Sequence No. 23). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pID1 (FIG. 10 and Sequence No.47). Upon growing the colony in Luria Bertani (LB) medium, a SDS-PAGE of the cellular lysate was done. As a result a 25 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the N-terminal region from the MDH protein and the DENe protein fragment from the DEN-4 virus. The protein was recognized in Immunoblotting by polyclonal antibodies (PA) anti-DEN4 contained in the HMAF. This protein was denominated PID1 (Sequence No.48).

Example 38 Purification of the Protein PID1

[0146] The biomass obtained from the E. coli strain transformed with pID1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained preponderantly as insoluble form associated to the pellet of the cellular disruption. From the pellet, the protein was extracted with urea 6 M and the supernatant, containing the PID1 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The protein was eluted with Imidazolee 60 mM and the obtained volume was loaded onto a G-25 column to finally obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.

Example 39 Antigenic Characterization of PID1

[0147] The purified fraction of PID1 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 46).

[0148] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN4. The recognition by HMAF against the other serotypes was lower than in the case of serotype 4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN4 was measured, achieving a substantial signal in both cases by Dot blotting Western blotting. TABLE 46 Reactivity of PID1 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PID1 HMAF DEN-1 − HMAF DEN-2 − HMAF DEN-3 + HMAF DEN-4 ++ HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 40 Characterization of the Antibody Response Generated by PID1

[0149] A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PID1 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 47 and table 50). In addition, the HI assay was done and only positive titers were found against DEN-4 (table 48 and table 50). Finally, the in vitro neutralization assay was done and neutralization titers of 1:320 against DEN-4 were obtained. However, no neutralization of the viral infection was found against the rest of the serotypes (table 49 and table 50). These results indicate the high serotype-specificity of the antibodies elicited by PID1. TABLE 47 Antibody titers against DEN-4 from the sera obtained upon immunization of mice with PID1. Titers Titers anti-DEN-4 anti-DEN-4 Mouse PID1 PBS Control (−) 1 1/128 000 <1:100 2 1/128 000 <1:100 3 1/64 000 <1:100 4 1/64 000 <1:100 5 1/128 000 <1:100 6 1/32 000 <1:100 7 1/128 000 <1:100 8 1/32 000 <1:100 9 1/128 000 <1:100 10 1/128 000 <1:100

[0150] TABLE 48 Titers by HI of the sera from the animals immunized with PID1. Titers by HI* Titers by HI anti-DEN-4 anti-DEN-4 Mouse PID1 PBS C(−) 1 1:320 <1:5 2 1:320 <1:5 3 1:640 <1:5 4 1:40 <1:5 5 <1/5 <1:5 6 1:320 <1:5 7 1:640 <1:5 8 1:640 <1:5 9 1:40 <1:5 10 1:320 <1:5

[0151] TABLE 49 Viral neutralization assay with the sera of animals immunized with PID1. Neutralizing Neutralizing titers titers anti-DEN-4 anti-DEN-4 Mouse PID1 PBS C(−) 1 1:320 <1:5 2 1:80 <1:5 3 1:320 <1:5 4 1:320 <1:5 5 1:160 <1:5 6 1:320 <1:5 7 1:320 <1:5 8 1:320 <1:5 9 1:160 <1:5 10 1:40 <1:5

[0152] TABLE 50 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PID1. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1 (PID1) <1/100 <1/100 <1/100  1:64 000 2(PID1) <1/100 <1/100 <1/100 >1:128 000 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PID1 <1/5 <1/5 <1/5 >1:320 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PID1) <1:5 <1:5 <1:5 1:160 2(PID1) <1:5 <1:5 <1:5 1:320

Example 41 Obtención de pID2

[0153] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-4 virus (Sequence No.46) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.16 and Sequence No.18 from a DEN-4 viral strain (Zhao B., Mackow E. R., Buckler-White A. J., Markoff L., Chancock R. M., Lai C. -J., Makino Y. Cloning full-length Dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 1986.155:77-88. The vector was created by digestion of the pM84 His plasmid with Xba I/Eco RI, which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines (Sequence No. 26). This digestion permits the insertion of the amplified fragment by PCR within the coding region for a structural domain of the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 were transformed with the selected clone, called pID2 (FIG. 11 and Sequence No.49). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-4 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN4 and was denominated PID2 (Sequence No.50).

Example 42 Purification of the PID2 Protein

[0154] The biomass obtained from the E. coli strain transformed with pID2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. The protein associated to the insoluble fraction was extracted using Urea 6 M, and the supernatant, containing the PID2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The column was washed with Imidazolee 30 mM and the protein was eluted with Imidazole 250 mM. Finally, the pure fraction was loaded onto a G-25 column to obtain the protein in the formulation (PBS). This preparation was used for immunological studies.

Example 43 Caracterización antigénica de PID2

[0155] The purified fraction of PID2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 51).

[0156] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN4 (higher than those obtained with PID1). The recognition by HMAF against the other serotypes was lower than in the case of serotype 4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against five human sera of high titers and three of low titers against DEN-3 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting. TABLE 51 Reactivity of PID2 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PID2 HMAF DEN-1 − HMAF DEN-2 − HMAF DEN-3 + HMAF DEN-4 +++ HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 44 Characterization of the Antibody Response Generated by PID2

[0157] A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PID2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-4 were obtained while, no reactivity was obtained against the rest of the serotypes (table 52 and table 55). In addition, the HI assay was done and only positive titers were found against DEN-4 (table 53 and table 55). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-4 were obtained (table 54). No neutralization of the viral infection was found against the rest of the serotypes (table 55). TABLE 52 Antibody titers against DEN-4 from the sera obtained upon immunization of mice with PID2. Titers Titers anti-DEN-4 anti-DEN-4 Mouse PID2 PBSControl (−) 1  1:64000 <1:100 2 >1:128 000 <1:100 3  1:128 000 <1:100 4 >1:128 000 <1:100 5  1:64000 <1:100 6 >1:128 000 <1:100 7 >1:128 000 <1:100 8 >1:128 000 <1:100 9 >1:128 000 <1:100 10  1:128 000 <1:100

[0158] TABLE 53 Titers by HI of the sera from the animals immunized with PID2. Titers by HI* Titers by HI anti-DEN-4 anti-DEN-4 Mouse (PID2) PBS C(−) 1 1:320 <1:5 2 1:320 <1:5 3 1:640 <1:5 4 1:40 <1:5 5 <1/5 <1:5 6 1:320 <1:5 7 1:640 <1:5 8 1:640 <1:5 9 1:40 <1:5 10 1:320 <1:5

[0159] TABLE 54 Viral neutralization assay with the sera of animals immunized with PID2. Neutralizing Neutralizing titers titers anti-DEN-4 anti-DEN-4 Mouse PID2 PBS C(−) 1 >1:1280 <1:5 2 >1:1280 <1:5 3 >1:1280 <1:5 4 >1:1280 <1:5 5  1:640 <1:5 6 >1:1280 <1:5 7 >1:1280 <1:5 8 >1:1280 <1:5 9  1:640 <1:5 10 >1:1280 <1:5

[0160] TABLE 55 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PID2. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1(PID2) <1/100 <1/100 <1/100 >1:128 000 2(PID2) <1/100 <1/100 <1/100  1:64 000 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PID2 <1/5 <1/5 <1/5 >1/320 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PID2) <1:5 <1:5 <1:5 >1:1280 2(PID2) <1:5 <1:5 <1:5 >1:1280

Example 45 Obtaining of pID3

[0161] The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN4 virus (Sequence No.46) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.19 and Sequence No.20 from the DEN4 viral strain (Zhao B., Mackow E. R., Buckler-White A. J., Markoff L., Chancock R. M., Lai C. -J., Makino Y. Cloning full-length Dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 1986.155:77-88).

[0162] The vector was created by digestion of the pD4 plasmid with Bam HI/Bam HI which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines without stop codon (Sequence No.29). This digestion permits the fusion of the amplified fragment by PCR after the C-terminal region for the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pID3 (FIG. 12 and Sequence No.51). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN4 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-4 and was denominated PID3 (Sequence No.52).

Example 46 Purificación de la proteina PID3

[0163] The biomass obtained from the E. coli strain transformed with pID3 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the insoluble fraction, the protein was extracted using Urea 6 M, and the supernatant, containing the PID3 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-Sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. The colum was washed with Imidazolee 45 mM and the protein was eluted with Imidazole 200 mM. Finally, the pure fraction of the protein was loaded onto a G-25 column to obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.

Example 47 Antigenic Characterization of PID3

[0164] The purified fraction of PID3 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 56).

[0165] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN4. The recognition by HMAF against the other serotypes was lower than in the case of serotype 4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN4 was measured, achieving a substantial signal in both cases by Dot blotting and Western blotting. TABLE 56 Reactivity of PID3 protein to monoclonal and polyclonal antibodies. Abs** Specificity*** PID3 HMAF DEN-1 − HMAF DEN-2 − HMAF DEN-3 + HMAF DEN-4 +++ HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 48 Characterization of the Antibody Response Generated by PID3

[0166] A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PAZ3 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-4 were obtained while, no reactivity was obtained against the rest of the serotypes (table 57 and table 60). In addition, the HI assay was done and only positive titers were found against DEN4 (table 58 and table 60). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN4 were obtained (table 59). No neutralization of the viral infection was found against the rest of the serotypes (table 60). TABLE 57 Antibody titers against DEN-4 from the sera obtained upon immunization of mice with PID3. Titers Titers anti-DEN-4 anti-DEN-4 Mouse (PID3) PBSControl (−) 1 >1:128 000 <1:100 2 >1:128 000 <1:100 3 >1:128 000 <1:100 4  1:64000 <1:100 5  1:64 000 <1:100 6 >1:128 000 <1:100 7  1:128 000 <1:100 8 >1:128 000 <1:100 9  1:128 000 <1:100 10 >1:128 000 <1:100

[0167] TABLE 58 Titers by HI of the sera from the animals immunized with PID3. Titers by HI* Titers by HI anti-DEN-4 anti-DEN-4 Mouse PID3 PBS C(−) 1 >1:640 <1:5 2  1:320 <1:5 3 >1:640 <1:5 4 >1:640 <1:5 5 <1:5 <1:5 6 >1:640 <1:5 7  1:320 <1:5 8 >1:640 <1:5 9  1:320 <1:5 10 >1:640 <1:5

[0168] TABLE 59 Viral neutralization assay with the sera of animals immunized with PID3. Neutralizing Neutralizing titers* titers anti-DEN-4 anti-DEN-4 Mouse PID3 PBS C(−) 1 >1:1280 <1:5 2 >1:1280 <1:5 3 >1:1280 <1:5 4 >1:1280 <1:5 5 >1:1280 <1:5 6 >1:1280 <1:5 7 >1:1280 <1:5 8  1:640 <1:5 9 >1:1280 <1:5 10 >1:1280 <1:5

[0169] TABLE 60 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PID3. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1(PID3) <1/100 <1/100 <1/100 >1:128 000 2(PID3) <1/100 <1/100 <1/100  1:128 000 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PID3 <1/5 <1/5 <1/5 >1/320 Neutralizing Neutralizing Neutralizing Neutralizing Mixture of titers*** titers titers titers sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PID3) <1:5 <1:5 <1:5 >1:1280 2(PID3) <1:5 <1:5 <1:5 >1/1280

Example 49 Obtaining of pD4D2

[0170] The nucleotide sequence that codifies for the amino acids 286 to 426 of the E protein from the DEN-4 virus (Sequence No.46) was amplified with the oligonuclotides identified in the sequencing list as Sequence No.16 and Sequence No.21 from the viral strain of DEN-4 (Zhao B., Mackow E. R., Buckler-White A. J., Markoff L., Chancock R. M., Lai C. -J., Makino Y. Cloning full-length Dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 1986. 155:77-88).

[0171] The vector was created by digestion Xba/Xba I of the pLL3 plasmid, which contains the MDH gene plus a sequence of 6 histidines in the 3′ region of the gene and the sequence of the E fragment from DEN-2 in the 3′ end. As result, two regions of the E protein from serotypes 2 and 4 were obtained, fused to the same MDH gene. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 were transformed with the selected clone, called pD4D2 (FIG. 13 and Sequence No.53). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 110 kDa band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the MDH protein and the two fragments of the DENe protein from Dengue virus. This protein was recognized by polyclonal antibodies anti-DEN-2 and anti-DEN-4 contained in the HMAF. This protein was denominated PD4D2 (Sequence No.54).

Example 50 Purification of the PD4D2 Protein

[0172] The biomass obtained from the E. coli strain transformed with pD4D2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained either soluble or insoluble. From the pellet the protein was extracted with urea 6 M and the supernatant, containing the PD4D2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu⁺⁺ ions. A washing step was done with Imidazolee 30 mM and the protein was eluted with Imidazole 250 mM. Finally, the pure preparation was loaded onto a G-25 column to obtain the protein in the formulation buffer and use it for immunological studies.

Example 51 Antigenic Characterization of PD4D2

[0173] The purified fraction of PD4D2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 61).

[0174] The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-2 and anti-DEN-4. The recognition of the two other serotypes was less than for the case of serotypes 2 and 4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. On the other hand, the Mab 3H5 had reactivity indeed, similar to that obtained for PLL2 and PLL3.

[0175] Finally, the reactivity against human sera of high and low titers against DEN-2 and DEN-4 was measured, achieving a substantial signal for both cases by Western blotting and Dot blotting. TABLE 61 Reactivity of PD4D2 protein against monoclonal and polyclonal antibodies. Abs** Specificity*** PD4D2 HMAF DEN-1 − HMAF DEN-2 +++ HMAF DEN-3 − HMAF DEN-4 +++ HMAF EEE − HMAF YFV − HMAF SLV − Mab 3H5 NT −

Example 52 Characterization of the Antibody Response Generated by PD4D2

[0176] A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PD4D2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-2 and DEN4 were obtained while, no reactivity was obtained against the rest of the serotypes (table 62 and table 65). In addition, the hemagglutination inhibition assay (HI) was done and only positive titers were found against DEN-2 and DEN-4 (table 63 and table 65). Finally, the in vitro neutralization assay was done and neutralization titers of >1:1280 against DEN-2 and >1:1280 against DEN-4 were obtained (table 64). No neutralization of the viral infection was found against the rest of the serotypes (table 65). TABLE 62 Antibody titers against DEN-2 and DEN-4 from the sera obtained upon immunization of mice with PD4D2. Titers by ELISA Titers by ELISA (PD4D2) PBS C(−) Mouse Anti-DEN-4 Anti-DEN-2 Anti-DEN-4 Anti-DEN-2 1 >1:128 000 >1:128 000 <1:100 <1:100 2  1:128 000  1:128 000 <1:100 <1:100 3 >1:128 000 >1:128 000 <1:100 <1:100 4 >1:128 000 >1:128 000 <1:100 <1:100 5  1:64 000 >1:128 000 <1:100 <1:100 6 >1:128 000  1:128 000 <1:100 <1:100 7  1:64 000 >1:128 000 <1:100 <1:100 8 >1:128 000 >1:128 000 <1:100 <1:100 9 >1:128 000  1:128 000 <1:100 <1:100 10  1:128 000 >1:128 000 <1:100 <1:100

[0177] TABLE 63 Titers by HI of the sera from the animals immunized with PD4D2. Titers by HI (PD4D2) Titers by HI PBS C(−) Mouse Anti-DEN-4 Anti-DEN-2 Anti-DEN-4 Anti-DEN-2 1 >1:640 >1:640 <1:5 <1:5 2 >1:640 >1:640 <1:5 <1:5 3 >1:640  1:320 <1:5 <1:5 4 >1:640 >1:640 <1:5 <1:5 5  1:320  1:640 <1:5 <1:5 6 >1:640 >1:640 <1:5 <1:5 7 >1:640 >1:640 <1:5 <1:5 8  1:320  1:320 <1:5 <1:5 9  1:320  1:320 <1:5 <1:5 10 >1:640 >1:640 <1:5 <1:5

[0178] TABLE 64 Viral neutralization assay with the sera of animals immunized with PD4D2. Neutralizing titers* Neutralizing titers (PD4D2) PBS C(−) Mouse Anti-DEN-4 Anti-DEN-2 Anti-DEN-4 Anti-DEN-2 1 >1:1280 >1:1280 <1:5 <1:5 2 >1:1280 >1:1280 <1:5 <1:5 3  1:1280  1:1280 <1:5 <1:5 4 >1:1280 >1:1280 <1:5 <1:5 5  1:640  1:1280 <1:5 <1:5 6 >1:1280 >1:1280 <1:5 <1:5 7 >1:1280 >1:1280 <1:5 <1:5 8  1:640 >1:1280 <1:5 <1:5 9 >1:1280 >1:1280 <1:5 <1:5 10 >1:1280 >1:1280 <1:5 <1:5

[0179] TABLE 65 Cross-reactivity assay against all the viral serotypes by ELISA, HI and viral neutralization with the sera of animals immunized with PD4D2. Mixture ELISA ELISA ELISA ELISA of sera* (anti-DEN-1) (anti DEN-2) (anti-DEN-3) (anti-DEN-4) 1(PD4D2) <1/100 >1:128 000 <1/100  1:64 000 2(PD4D2) <1/100 >1:128 000 <1/100 >1:128 000 Mixture HI** HI HI HI of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 PD4D2 <1:5 >1:320 <1:5 >1:320 Neutralizing Neutralizing Neutralizing Neutralizing Mixture titers*** titers titers titers of sera* anti-DEN-1 anti-DEN-2 anti-DEN-3 anti-DEN-4 1(PD4D2) <1:5 >1:1280 <1:5 >1:1280 2(PD4D2) <1:5 >1:1280 <1:5 >1:1280

Example 53 Protection Assay

[0180] For the evaluation of the protection conferred to mice immunized with all the assayed variants upon challenge with homologous lethal DEN, 15 mice of each group were used. Each animal received one dose of 100 LD₅₀ of lethal DEN by intracraneal inoculation and they were observed during 21 days to study the percentage of lethality. As positive controls, groups of 15 mice immunized with the four viral preparations (DEN-1, DEN-2, DEN-3 and DEN-4) were used. All the mice of these control groups survived while mice from the negative control group become sick between 7-11 days after challenge; therefore, achieving a 100% of mortality. Finally, groups immunized with the fusion proteins under study had between an 80% and 100% of protection and in all the cases, significant differences with respect to the control group were found (table 66). TABLE 66 Percentage of survival in mice immunized with the protein variants assayed upon challenge with the homologous lethal Dengue virus. Percentage of Immunogen survival* PBS 0 DEN-1 100 DEN-2 100 DEN-3 100 DEN-4 100 PLL1 86 PLL2 100 PLL3 100 PLH1 80 PLH2 100 PLH3 100 PAZ1 80 PAZ2 100 PAZ3 100 PID1 86 PID2 100 PID3 100 PD4D2 100 (DEN-4) 100 (DEN-2)

Example 54 Lymphoproliferative Response

[0181] Animals from different groups immunized with the chimeric proteins containing the E fragment from DEN-2 (PLL1, PLL2 and PLL3), and a placebo group, were sacrified 15 days after the last dose. Then, the spleen of the animals was harvested and the lymphoproliferative response against the four serotypes of Dengue virus was studied. Table 67 shows the results of the stimulation indexes obtained, which demonstrate that a serotype specific response was achieved. TABLE 67 Stimulation indexes, against the four Dengue viral serotypes, of lymphocytes from mice immunized with PLL1, PLL2 and PLL3. PLL1 PLL2 PLL3 C(−) DEN-1   1.3* 1.0 0.8 1.2 DEN-2 12.5 10.3 8.9 1.4 DEN-3 1.0 1.6 1.8 1.4 DEN-4 1.7 1.5 1.7 1.1 Control Antigen 1.1 1.0 1.3 0.9 PHA** 13.3 16.5 11.1 12.0

[0182]

1 54 1 25 DNA Escherichia coli primer_bind (1)..(25) Sequence of the Xba-I primer for the amplification of DENe-2 fragment 1 cttctagaca ggctgcgcat ggaca 25 2 29 DNA Escherichia coli primer_bind (1)..(29) Sequence of the Bam-HI primer for the amplification of DENe-2 fragment 2 gtggatcctt accctcccag gcttccaaa 29 3 25 DNA Escherichia coli primer_bind (1)..(25) Sequence of the Eco-RI primer for the amplification of DENe-2 fragment 3 atgaattcac gcctcccaga gatcc 25 4 21 DNA Escherichia coli primer_bind (1)..(21) Sequence of the Bam-HI primer for the amplification of DENe-2 fragment 4 cttggatcca ggctgagaat g 21 5 31 DNA Escherichia coli primer_bind (1)..(31) Sequence of the Bam-HI primer for the amplification of DENe-2 fragment 5 gaggatcctt aaccacccag agacccaaaa t 31 6 25 DNA Escherichia coli primer_bind (1)..(25) Sequence of the Xba-I primer for the amplification of DENe-1 fragment 6 cttctagaca ggctcaaaat ggata 25 7 28 DNA Escherichia coli primer_bind (1)..(28) Sequence of the Bam-HI primer for the amplification of DENe-1 fragment 7 gaggatcctt acccgccaat agaaccga 28 8 25 DNA Escherichia coli primer_bind (1)..(25) Sequence of the Eco-RI primer for the amplification of DENe-1 fragment 8 acgaattcac ccctcctata gatcc 25 9 24 DNA Escherichia coli primer_bind (1)..(24) Sequence of the Bam-HI primer for the amplification of DENe-1 fragment 9 acaccttgga tccagactaa aaat 24 10 26 DNA Escherichia coli primer_bind (1)..(26) Sequence of the Bam-HI primer for the amplification of DENe-1 fragment 10 ccggatccgt gaattaccca cctata 26 11 29 DNA Escherichia coli primer_bind (1)..(29) Sequence of the Xba-I primer for the amplification of DENe-3 fragment 11 tttctagata gactcaagat ggacaaatt 29 12 28 DNA Escherichia coli primer_bind (1)..(28) Sequence of the Bam-HI primer for the amplification of DENe-3 fragment 12 gaggatcctt aaccacccac tgaaccaa 28 13 25 DNA Escherichia coli primer_bind (1)..(25) Sequence of the Eco-RI primer for the amplification of DENe-3 fragment 13 aagaattcac accacccaca gatcc 25 14 23 DNA Escherichia coli primer_bind (1)..(23) Sequence of the Bam-HI primer for the amplification of DENe-3 fragment 14 acttaggatc cagactcaag atg 23 15 26 DNA Escherichia coli primer_bind (1)..(26) Sequence of the Bam-HI primer for the amplification of DENe-3 fragment 15 gaggatcctt aaccacccac tgaacc 26 16 30 DNA Escherichia coli primer_bind (1)..(30) Sequence of the Xba-I primer for the amplification of DENe-4 fragment 16 cttctagaca aagtgcgtat ggagaaattg 30 17 28 DNA Escherichia coli primer_bind (1)..(28) Sequence of the Bam-HI primer for the amplification of DENe-4 fragment 17 gaggatcctt aaccaccaac agaaccaa 28 18 25 DNA Escherichia coli primer_bind (1)..(25) Sequence of the Eco-RI primer for the amplification of DENe-4 fragment 18 atgaattcag tccaccaacg ctacc 25 19 27 DNA Escherichia coli primer_bind (1)..(27) Sequence of the Bam-HI primer for the amplification of DENe-4 fragment 19 ggccatctag gatccaaagt gcgtatg 27 20 28 DNA Escherichia coli primer_bind (1)..(28) Sequence of the Bam-HI primer for the amplification of DENe-4 fragment 20 gaggatcctt agccaccaac cgaaccaa 28 21 26 DNA Escherichia coli primer_bind (1)..(26) Sequence of the primer Xba-I for the amplification of DENe-4 fragment 21 attctagaag accaccaacg gaacca 26 22 429 DNA Escherichia coli gene (1)..(426) Nucleotidic sequence coding for the aminoacids 286 to 426 of DEN-2 envelope protein 22 gacaggctga gaatggacaa actacagctc aaaggaatgt catactctat gtgtacagga 60 aagtttaaaa ttgtgaagga aatagcagaa acacaacatg gaacaatagt tatcagagta 120 caatatgaag gggacggctc tccatgtaag atcccttttg agataatgga tttggaaaaa 180 agacacgtct taggtcgcct gattacagtt aacccgatcg taacagaaaa agatagccca 240 gtcaacatag aagcagaacc tccattcgga gacagctaca tcatcatagg agtagagccg 300 ggacaattga aactcaactg gtttaagaaa ggaagttcca tcggccaaat gtttgagaca 360 acaatgagag gagcgaagag aatggccatt ttaggtgaca cagcctggga ttttggatcc 420 ctgggagga 429 23 168 DNA Escherichia coli gene (1)..(168) Nucleotidic sequence coding for the first 45 aminoacids of the MDH. 23 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagac 168 24 603 DNA Escherichia coli gene (1)..(603) Nucleotidic sequence coding for the quimeric protein in the plasmid pLL1 24 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagacag gctgcgcatg 180 gacaaactac agctcaaagg aatgtcatac tctatgtgta caggaaagtt taaaattgtg 240 aaggaaatag cagaaacaca acatggaaca atagttatca gagtacaata tgaaggggac 300 ggctctccat gtaagatccc ttttgagata atggatttgg aaaaaagaca cgtcttaggt 360 cgcctgatta cagttaaccc gatcgtaaca gaaaaagata gcccagtcaa catagaagca 420 gaacctccat tcggagacag ctacatcatc ataggagtag agccgggaca attgaaactc 480 aactggttta agaaaggaag ttccatcggc caaatgtttg agacaacaat gagaggagcg 540 aagagaatgg ccattttagg tgacacagcc tgggattttg gaagcctggg agggtaagga 600 tcc 603 25 195 PRT Escherichia coli CHAIN (1)..(195) Aminoacidic sequence of the PLL1 protein 25 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Leu Asp Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly 50 55 60 Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Ile Val Lys Glu Ile 65 70 75 80 Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly 85 90 95 Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys 100 105 110 Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu 115 120 125 Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser 130 135 140 Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe 145 150 155 160 Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly 165 170 175 Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 180 185 190 Leu Gly Gly 195 26 1851 DNA Escherichia coli gene (1)..(1851) Nucleotidic sequence of the MDH in the plasmid pM84 His. 26 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagaa 180 gaactagtgg atcccccggg ctgcaggaat tcgatgaatt cgatggacgt acctgctgaa 240 gttgcaggcg tagtcaaaga agttaaagtt aaagtcggcg acaaaatctc tgaaggtggt 300 ttgattgtcg tcgttgaagc tgaaggcacg gcagccgctc ctaaagccga agcggctgcc 360 gccccggcgc aagaagcccc taaagctgcc gctcctgctc cgcaagccgc gcaattcggc 420 ggttctgccg atgccgagta cgacgtggtc gtattgggtg gcggtcccgg cggttactcc 480 gctgcatttg ccgctgccga tgaaggcttg aaagtcgcca tcgtcgaacg ttacaaaact 540 ttgggcggcg tttgcctgaa cgtcggctgt atcccttcca aagccttgtt gcacaatgcc 600 gccgttatcg acgaagtgcg ccacttggct gccaacggta tcaaataccc cgagccggaa 660 ctcgacatcg atatgcttcg cgcctacaaa gacggcgtag tttcccgcct cacgggcggt 720 ttggcaggta tggcgaaaag ccgtaaagtg gacgttatcc aaggcgacgg gcaattctta 780 gatccgcacc acttggaagt gtcgctgact gccggcgacg cgtacgaaca ggcagcccct 840 accggcgaga aaaaaatcgt tgccttcaaa aactgtatca ttgcagcagg cagccgcgta 900 accaaactgc ctttcattcc tgaagatccg cgcatcatcg attccagcgg cgcattggct 960 ctgaaagaag taccgggcaa actgctgatt atcggcggcg gcattatcgg cctcgagatg 1020 ggtacggttt acagcacgct gggttcgcgt ttggatgtgg ttgaaatgat ggacggcctg 1080 atgcaaggcg cagaccgcga tttggtaaaa gtatggcaaa aacaaaacga ataccgtttt 1140 gacaacatta tggtcaacac caaaaccgtt gcagttgagc cgaaagaaga cggcgtttac 1200 gttacctttg aaggcgcgaa cgcgcctaaa gagccgcaac gctacgatgc cgtattggtt 1260 gccgccggcc gcgcgcccaa cggcaaactc atcagcgcgg aaaaagcagg cgttgccgta 1320 accgatcgcg gcttcatcga agtggacaaa caaatgcgta ccaatgtgcc gcacatctac 1380 gccatcggcg acatcgtcgg tcagccgatg ttggcgcaca aagccgttca cgaaggccac 1440 gttgccgccg aaaactgcgc cggccacaaa gcctacttcg acgcacgcgt gattccgggc 1500 gttgcctaca cttcccccga agtggcgtgg gtgggcgaaa ccgaactgtc cgccaaagcc 1560 tccggccgca aaatcaccaa agccaacttc ccgtgggcgg cttccggccg tgcgattgcc 1620 aacggttgcg acaagccgtt taccaagctg atttttgatg ccgaaaccgg ccgcatcatc 1680 ggcggcggca ttgtcggtcc gaacggtggc gatatgatcg gcgaagtctg ccttgccatc 1740 gaaatgggct gcgacgcggc agacatcggc aaaaccatcc acccgcaccc gggcgaatcc 1800 atcggtatgg cggcggaagt ggcattgggt acttgtaccg acaaaaaaaa a 1851 27 2253 DNA Escherichia coli gene (1)..(2253) Nucleotidic sequence of the quimeric protein in the plasmid pLL2. 27 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagac 180 aggctgcgca tggacaaact acagctcaaa ggaatgtcat actctatgtg tacaggaaag 240 tttaaaattg tgaaggaaat agcagaaaca caacatggaa caatagttat cagagtacaa 300 tatgaagggg acggctctcc atgtaagatc ccttttgaga taatggattt ggaaaaaaga 360 cacgtcttag gtcgcctgat tacagttaac ccgatcgtaa cagaaaaaga tagcccagtc 420 aacatagaag cagaacctcc attcggagac agctacatca tcataggagt agagccggga 480 caattgaaac tcaactggtt taagaaagga agttccatcg gccaaatgtt tgagacaaca 540 atgagaggag cgaagagaat ggccatttta ggtgacacag cctgggattt tggatctctg 600 ggaggcgtga attcgatgaa ttcgatggac gtacctgctg aagttgcagg cgtagtcaaa 660 gaagttaaag ttaaagtcgg cgacaaaatc tctgaaggtg gtttgattgt cgtcgttgaa 720 gctgaaggca cggcagccgc tcctaaagcc gaagcggctg ccgccccggc gcaagaagcc 780 cctaaagctg ccgctcctgc tccgcaagcc gcgcaattcg gcggttctgc cgatgccgag 840 tacgacgtgg tcgtattggg tggcggtccc ggcggttact ccgctgcatt tgccgctgcc 900 gatgaaggct tgaaagtcgc catcgtcgaa cgttacaaaa ctttgggcgg cgtttgcctg 960 aacgtcggct gtatcccttc caaagccttg ttgcacaatg ccgccgttat cgacgaagtg 1020 cgccacttgg ctgccaacgg tatcaaatac cccgagccgg aactcgacat cgatatgctt 1080 cgcgcctaca aagacggcgt agtttcccgc ctcacgggcg gtttggcagg tatggcgaaa 1140 agccgtaaag tggacgttat ccaaggcgac gggcaattct tagatccgca ccacttggaa 1200 gtgtcgctga ctgccggcga cgcgtacgaa caggcagccc ctaccggcga gaaaaaaatc 1260 gttgccttca aaaactgtat cattgcagca ggcagccgcg taaccaaact gcctttcatt 1320 cctgaagatc cgcgcatcat cgattccagc ggcgcattgg ctctgaaaga agtaccgggc 1380 aaactgctga ttatcggcgg cggcattatc ggcctcgaga tgggtacggt ttacagcacg 1440 ctgggttcgc gtttggatgt ggttgaaatg atggacggcc tgatgcaagg cgcagaccgc 1500 gatttggtaa aagtatggca aaaacaaaac gaataccgtt ttgacaacat tatggtcaac 1560 accaaaaccg ttgcagttga gccgaaagaa gacggcgttt acgttacctt tgaaggcgcg 1620 aacgcgccta aagagccgca acgctacgat gccgtattgg ttgccgccgg ccgcgcgccc 1680 aacggcaaac tcatcagcgc ggaaaaagca ggcgttgccg taaccgatcg cggcttcatc 1740 gaagtggaca aacaaatgcg taccaatgtg ccgcacatct acgccatcgg cgacatcgtc 1800 ggtcagccga tgttggcgca caaagccgtt cacgaaggcc acgttgccgc cgaaaactgc 1860 gccggccaca aagcctactt cgacgcacgc gtgattccgg gcgttgccta cacttccccc 1920 gaagtggcgt gggtgggcga aaccgaactg tccgccaaag cctccggccg caaaatcacc 1980 aaagccaact tcccgtgggc ggcttccggc cgtgcgattg ccaacggttg cgacaagccg 2040 tttaccaagc tgatttttga tgccgaaacc ggccgcatca tcggcggcgg cattgtcggt 2100 ccgaacggtg gcgatatgat cggcgaagtc tgccttgcca tcgaaatggg ctgcgacgcg 2160 gcagacatcg gcaaaaccat ccacccgcac ccgggcgaat ccatcggtat ggcggcggaa 2220 gtggcattgg gtacttgtac cgacaaaaaa aaa 2253 28 748 PRT Escherichia coli CHAIN (1)..(748) Aminoacidic sequence of PLL2. 28 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Leu Asp Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly 50 55 60 Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Ile Val Lys Glu Ile 65 70 75 80 Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly 85 90 95 Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys 100 105 110 Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu 115 120 125 Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser 130 135 140 Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe 145 150 155 160 Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly 165 170 175 Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 180 185 190 Leu Gly Gly Val Asn Ser Met Asn Ser Met Asp Val Pro Ala Glu Val 195 200 205 Ala Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser 210 215 220 Glu Gly Gly Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala 225 230 235 240 Pro Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala 245 250 255 Ala Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala 260 265 270 Glu Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala 275 280 285 Ala Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg 290 295 300 Tyr Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser 305 310 315 320 Lys Ala Leu Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu 325 330 335 Ala Ala Asn Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met 340 345 350 Leu Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu 355 360 365 Ala Gly Met Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly 370 375 380 Gln Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp 385 390 395 400 Ala Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe 405 410 415 Lys Asn Cys Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe 420 425 430 Ile Pro Glu Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu 435 440 445 Lys Glu Val Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly 450 455 460 Leu Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val 465 470 475 480 Val Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val 485 490 495 Lys Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val 500 505 510 Asn Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val 515 520 525 Thr Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala 530 535 540 Val Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala 545 550 555 560 Glu Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp 565 570 575 Lys Gln Met Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile 580 585 590 Val Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val 595 600 605 Ala Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val 610 615 620 Ile Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu 625 630 635 640 Thr Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn 645 650 655 Phe Pro Trp Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys 660 665 670 Pro Phe Thr Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly 675 680 685 Gly Gly Ile Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys 690 695 700 Leu Ala Ile Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile 705 710 715 720 His Pro His Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu 725 730 735 Gly Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys 740 745 29 1821 DNA Escherichia coli gene (1)..(1821) Nucleotidic sequence of the MDH in the plasmid pD4 29 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct 180 gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa 240 ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg 300 gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa 360 ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt 420 tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac 480 aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac 540 aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag 600 ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg 660 ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa 720 ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca 780 gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc 840 cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca 900 ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc 960 gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac 1020 ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac 1080 cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc 1140 gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta 1200 ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt 1260 gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac 1320 atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa 1380 ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt 1440 ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc 1500 aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg 1560 attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc 1620 atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt 1680 gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc 1740 ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct 1800 ccgcaaaaga aaaaaggatc c 1821 30 2259 DNA Escherichia coli gene (1)..(2259) Nucleotidic sequence coding for the quimeric protein in the plasmid pLL3 30 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct 180 gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa 240 ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg 300 gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa 360 ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt 420 tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac 480 aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac 540 aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag 600 ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg 660 ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa 720 ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca 780 gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc 840 cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca 900 ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc 960 gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac 1020 ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac 1080 cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc 1140 gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta 1200 ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt 1260 gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac 1320 atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa 1380 ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt 1440 ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc 1500 aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg 1560 attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc 1620 atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt 1680 gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc 1740 ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct 1800 ccgcaaaaga aaaaaggatc cgacaggctg agaatggaca aactacagct caaaggaatg 1860 tcatactcta tgtgtacagg aaagtttaaa attgtgaagg aaatagcaga aacacaacat 1920 ggaacaatag ttatcagagt acaatatgaa ggggacggct ctccatgtaa gatccctttt 1980 gagataatgg atttggaaaa aagacacgtc ttaggtcgcc tgattacagt taacccgatc 2040 gtaacagaaa aagatagccc agtcaacata gaagcagaac ctccattcgg agacagctac 2100 atcatcatag gagtagagcc gggacaattg aaactcaact ggtttaagaa aggaagttcc 2160 atcggccaaa tgtttgagac aacaatgaga ggagcgaaga gaatggccat tttaggtgac 2220 acagcctggg attttgggtc tctgggtggt taaggatcc 2259 31 745 PRT Escherichia coli CHAIN (1)..(745) Aminoacidic sequence of PLL3 31 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val 50 55 60 Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 65 70 75 80 Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 85 90 95 Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 100 105 110 Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 115 120 125 Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 130 135 140 Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 145 150 155 160 Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu 165 170 175 Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn 180 185 190 Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala 195 200 205 Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met 210 215 220 Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu 225 230 235 240 Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu 245 250 255 Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys 260 265 270 Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 275 280 285 Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 290 295 300 Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 305 310 315 320 Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 325 330 335 Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 340 345 350 Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 355 360 365 Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 370 375 380 Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 385 390 395 400 Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala 405 410 415 Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met 420 425 430 Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln 435 440 445 Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu 450 455 460 Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly 465 470 475 480 Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu 485 490 495 Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp 500 505 510 Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 515 520 525 Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 530 535 540 Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 545 550 555 560 Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 565 570 575 Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys 580 585 590 Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Arg Leu Arg Met Asp 595 600 605 Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe 610 615 620 Lys Ile Val Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile 625 630 635 640 Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu 645 650 655 Ile Met Asp Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val 660 665 670 Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu Ala Glu 675 680 685 Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln 690 695 700 Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln Met Phe 705 710 715 720 Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala Ile Leu Gly Asp Thr 725 730 735 Ala Trp Asp Phe Gly Ser Leu Gly Gly 740 745 32 429 DNA Escherichia coli gene (1)..(429) Nucleotidic sequence coding for the aminoacids 286 to 426 of the DEN-1 envelope protein 32 agactaaaaa tggataaact gactttaaaa ggggtatcat atgtaatgtg cacagggtca 60 ttcaagttag agaaggaagt ggctgagacc cagcatggaa ctgttctagt gcaggttaaa 120 tacgaaggaa cagatgcacc atgcaagatc cccttctcgt cccaagatga gaaaggagta 180 acccagaatg ggagattgat aacagccaac cccatagtca ttgacaaaga aaaaccagtc 240 aacattgaag cggagccacc ttttggtgag agctatattg tggtaggagc aggtgaaaaa 300 gctttgaaac taagctggtt caagaaggga agcagtatag ggaaaatgtt tgaagcaact 360 gcccgtggag cacgaaggat ggccatcctg ggagacaccg catgggactt cggttctata 420 ggagggtaa 429 33 615 DNA Escherichia coli gene (1)..(615) Nucleotidic sequence coding for the quimeric protein in the plasmid pLH1 33 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagac 180 aggctcaaaa tggataaact gactttaaaa ggggtatcat atgtaatgtg cacagggtca 240 ttcaagttag agaaggaagt ggctgagacc cagcatggaa ctgttctagt gcaggttaaa 300 tacgaaggaa cagatgcacc atgcaagatc cccttctcgt cccaagatga gaaaggagta 360 acccagaatg ggagattgat aacagccaac cccatagtca ttgacaaaga aaaaccagtc 420 aacattgaag cggagccacc ttttggtgag agctatattg tggtaggagc aggtgaaaaa 480 gctttgaaac taagctggtt caagaaggga agcagtatag ggaaaatgtt tgaagcaact 540 gcccgtggag cacgaaggat ggccatcctg ggagacaccg catgggactt cggttctatt 600 ggcgggtaag gatcc 615 34 174 PRT Escherichia coli CHAIN (1)..(174) Aminoacidic sequence of the PLH1 34 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Leu Asp Phe Lys Leu Glu Lys Glu Val Ala Glu Thr Gln 50 55 60 His Gly Thr Val Leu Val Gln Val Lys Tyr Gln Gly Thr Asp Ala Pro 65 70 75 80 Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly Val Thr Gln Asn 85 90 95 Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp Lys Glu Lys Pro Val 100 105 110 Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu Ser Tyr Ile Val Val Gly 115 120 125 Ala Gly Glu Lys Ala Leu Lys Gln Cys Trp Phe Lys Lys Gly Ser Ser 130 135 140 Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala 145 150 155 160 Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Ile Gly Gly 165 170 35 2253 DNA Escherichia coli gene (1)..(2253) Nucleotidic sequence coding for the quimeric protein in the plasmid pLH2 35 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagac 180 aggctcaaaa tggataaact gactttaaaa ggggtatcat atgtaatgtg cacagggtca 240 ttcaagttag agaaggaagt ggctgagacc cagcatggaa ctgttctagt gcaggttaaa 300 tacgaaggaa cagatgcacc atgcaagatc cccttctcgt cccaagatga gaaaggagta 360 acccagaatg ggagattgat aacagccaac cccatagtca ttgacaaaga aaaaccagtc 420 aacattgaag cggagccacc ttttggtgag agctatattg tggtaggagc aggtgaaaaa 480 gctttgaaac taagctggtt caagaaggga agcagtatag ggaaaatgtt tgaagcaact 540 gcccgtggag cacgaaggat ggccatcctg ggagacaccg catgggactt cggatctata 600 ggaggggtga attcgatgaa ttcgatggac gtacctgctg aagttgcagg cgtagtcaaa 660 gaagttaaag ttaaagtcgg cgacaaaatc tctgaaggtg gtttgattgt cgtcgttgaa 720 gctgaaggca cggcagccgc tcctaaagcc gaagcggctg ccgccccggc gcaagaagcc 780 cctaaagctg ccgctcctgc tccgcaagcc gcgcaattcg gcggttctgc cgatgccgag 840 tacgacgtgg tcgtattggg tggcggtccc ggcggttact ccgctgcatt tgccgctgcc 900 gatgaaggct tgaaagtcgc catcgtcgaa cgttacaaaa ctttgggcgg cgtttgcctg 960 aacgtcggct gtatcccttc caaagccttg ttgcacaatg ccgccgttat cgacgaagtg 1020 cgccacttgg ctgccaacgg tatcaaatac cccgagccgg aactcgacat cgatatgctt 1080 cgcgcctaca aagacggcgt agtttcccgc ctcacgggcg gtttggcagg tatggcgaaa 1140 agccgtaaag tggacgttat ccaaggcgac gggcaattct tagatccgca ccacttggaa 1200 gtgtcgctga ctgccggcga cgcgtacgaa caggcagccc ctaccggcga gaaaaaaatc 1260 gttgccttca aaaactgtat cattgcagca ggcagccgcg taaccaaact gcctttcatt 1320 cctgaagatc cgcgcatcat cgattccagc ggcgcattgg ctctgaaaga agtaccgggc 1380 aaactgctga ttatcggcgg cggcattatc ggcctcgaga tgggtacggt ttacagcacg 1440 ctgggttcgc gtttggatgt ggttgaaatg atggacggcc tgatgcaagg cgcagaccgc 1500 gatttggtaa aagtatggca aaaacaaaac gaataccgtt ttgacaacat tatggtcaac 1560 accaaaaccg ttgcagttga gccgaaagaa gacggcgttt acgttacctt tgaaggcgcg 1620 aacgcgccta aagagccgca acgctacgat gccgtattgg ttgccgccgg ccgcgcgccc 1680 aacggcaaac tcatcagcgc ggaaaaagca ggcgttgccg taaccgatcg cggcttcatc 1740 gaagtggaca aacaaatgcg taccaatgtg ccgcacatct acgccatcgg cgacatcgtc 1800 ggtcagccga tgttggcgca caaagccgtt cacgaaggcc acgttgccgc cgaaaactgc 1860 gccggccaca aagcctactt cgacgcacgc gtgattccgg gcgttgccta cacttccccc 1920 gaagtggcgt gggtgggcga aaccgaactg tccgccaaag cctccggccg caaaatcacc 1980 aaagccaact tcccgtgggc ggcttccggc cgtgcgattg ccaacggttg cgacaagccg 2040 tttaccaagc tgatttttga tgccgaaacc ggccgcatca tcggcggcgg cattgtcggt 2100 ccgaacggtg gcgatatgat cggcgaagtc tgccttgcca tcgaaatggg ctgcgacgcg 2160 gcagacatcg gcaaaaccat ccacccgcac ccgggcgaat ccatcggtat ggcggcggaa 2220 gtggcattgg gtacttgtac cgacaaaaaa aaa 2253 36 727 PRT Escherichia coli CHAIN (1)..(727) Aminoacidic sequence of the PLH2 36 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Leu Asp Phe Lys Leu Glu Lys Glu Val Ala Glu Thr Gln 50 55 60 His Gly Thr Val Leu Val Gln Val Lys Tyr Gln Gly Thr Asp Ala Pro 65 70 75 80 Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly Val Thr Gln Asn 85 90 95 Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp Lys Glu Lys Pro Val 100 105 110 Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu Ser Tyr Ile Val Val Gly 115 120 125 Ala Gly Glu Lys Ala Leu Lys Gln Cys Trp Phe Lys Lys Gly Ser Ser 130 135 140 Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala 145 150 155 160 Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Ile Gly Gly Val Asn 165 170 175 Ser Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val Val Lys 180 185 190 Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly Leu Ile 195 200 205 Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala Glu Ala 210 215 220 Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro Ala Pro 225 230 235 240 Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp Val Val 245 250 255 Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala Ala Ala 260 265 270 Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr Leu Gly 275 280 285 Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu Leu His 290 295 300 Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn Gly Ile 305 310 315 320 Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala Tyr Lys 325 330 335 Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met Ala Lys 340 345 350 Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu Asp Pro 355 360 365 His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu Gln Ala 370 375 380 Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys Ile Ile 385 390 395 400 Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu Asp Pro 405 410 415 Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val Pro Gly 420 425 430 Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met Gly Thr 435 440 445 Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met Met Asp 450 455 460 Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp Gln Lys 465 470 475 480 Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys Thr Val 485 490 495 Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu Gly Ala 500 505 510 Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val Ala Ala 515 520 525 Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala Gly Val 530 535 540 Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met Arg Thr 545 550 555 560 Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln Pro Met 565 570 575 Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu Asn Cys 580 585 590 Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly Val Ala 595 600 605 Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu Ser Ala 610 615 620 Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp Ala Ala 625 630 635 640 Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr Lys Leu 645 650 655 Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile Val Gly 660 665 670 Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile Glu Met 675 680 685 Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His Pro Gly 690 695 700 Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys Thr Asp 705 710 715 720 Leu Pro Pro Gln Lys Lys Lys 725 37 2250 DNA Escherichia coli gene (1)..(2250) Nucleotidic sequence coding for the quimeric protein in the plasmid pLH3 37 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct 180 gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa 240 ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg 300 gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa 360 ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt 420 tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac 480 aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac 540 aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag 600 ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg 660 ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa 720 ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca 780 gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc 840 cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca 900 ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc 960 gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac 1020 ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac 1080 cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc 1140 gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta 1200 ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt 1260 gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac 1320 atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa 1380 ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt 1440 ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc 1500 aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg 1560 attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc 1620 atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt 1680 gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc 1740 ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct 1800 ccgcaaaaga aaaaaggatc cagactaaaa atggataaac tgactttaaa aggggtatca 1860 tatgtaatgt gcacagggtc attcaagtta gagaaggaag tggctgagac ccagcatgga 1920 actgttctag tgcaggttaa atacgaagga acagatgcac catgcaagat ccccttctcg 1980 tcccaagatg agaaaggagt aacccagaat gggagattga taacagccaa ccccatagtc 2040 attgacaaag aaaaaccagt caacattgaa gcggagccac cttttggtga gagctatatt 2100 gtggtaggag caggtgaaaa agctttgaaa ctaagctggt tcaagaaggg aagcagtata 2160 gggaaaatgt ttgaagcaac tgcccgtgga gcacgaagga tggccatcct gggagacacc 2220 gcatgggact tcggttctat aggtgggtaa 2250 38 724 PRT Escherichia coli CHAIN (1)..(724) Aminoacidic sequence of the PLH3 38 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val 50 55 60 Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 65 70 75 80 Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 85 90 95 Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 100 105 110 Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 115 120 125 Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 130 135 140 Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 145 150 155 160 Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu 165 170 175 Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn 180 185 190 Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala 195 200 205 Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met 210 215 220 Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu 225 230 235 240 Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu 245 250 255 Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys 260 265 270 Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 275 280 285 Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 290 295 300 Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 305 310 315 320 Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 325 330 335 Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 340 345 350 Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 355 360 365 Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 370 375 380 Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 385 390 395 400 Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala 405 410 415 Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met 420 425 430 Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln 435 440 445 Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu 450 455 460 Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly 465 470 475 480 Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu 485 490 495 Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp 500 505 510 Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 515 520 525 Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 530 535 540 Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 545 550 555 560 Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 565 570 575 Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys 580 585 590 Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Phe Lys Leu Glu Lys 595 600 605 Glu Val Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr 610 615 620 Gln Gly Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu 625 630 635 640 Lys Gly Val Thr Gln Asn Arg Leu Ile Thr Ala Asn Pro Ile Val Thr 645 650 655 Asp Lys Glu Lys Pro Val Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu 660 665 670 Ser Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Gln Cys Trp 675 680 685 Phe Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg 690 695 700 Gly Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly 705 710 715 720 Ser Ile Gly Gly 39 426 DNA Escherichia coli gene (1)..(426) Nucleotidic sequence coding for the aminoacids 286 to 426 of the DEN-3 envelope protein 39 agactcaaga tggacaaatt gaaactcaag gggatgagct atgcaatgtg cttgaatacc 60 tttgtgttga agaaagaagt ctccgaaacg cagcatggga caatactcat taaggttgag 120 tacaaagggg aagatgcacc ctgcaagatt cctttctcca cggaggatgg acaagggaaa 180 gctcacaatg gcagactgat cacagccaat ccagtggtga ccaagaagga ggagcctgtc 240 aacattgagg ctgaacctcc ttttggggaa agtaatatag taattggaat tggagacaaa 300 gccctgaaaa tcaactggta caggaaggga agctcgattg ggaagatgtt cgaggccact 360 gccagaggtg caaggcgcat ggccatcttg ggagacacag cctgggactt tggatcagtg 420 ggtggt 426 40 615 DNA Escherichia coli gene (1)..(615) Nucleotidic sequence coding for the quimeric protein in the plasmid pAZ1 40 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagat 180 agactcaaga tggacaaatt gaaactcaag gggatgagct atgcaatgtg cttgaatacc 240 tttgtgttga agaaagaagt ctccgaaacg cagcatggga caatactcat taaggttgag 300 tacaaagggg aagatgcacc ctgcaagatt cctttctcca cggaggatgg acaagggaaa 360 gctcacaatg gcagactgat cacagccaat ccagtggtga ccaagaagga ggagcctgtc 420 aacattgagg ctgaacctcc ttttggggaa agtaatatag taattggaat tggagacaaa 480 gccctgaaaa tcaactggta caggaaggga agctcgattg ggaagatgtt cgaggccact 540 gccagaggtg caaggcgcat ggccatcttg ggagacacag cctgggactt tggttcagtg 600 ggtggttaag gatcc 615 41 194 PRT Escherichia coli CHAIN (1)..(194) Aminoacidic sequence of the PAZ1 41 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Leu Asp Arg Leu Lys Met Asp Lys Leu Lys Leu Lys Gly 50 55 60 Met Ser Tyr Ala Met Cys Leu Asn Thr Phe Val Leu Lys Lys Glu Val 65 70 75 80 Ser Glu Thr His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu 85 90 95 Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys 100 105 110 Ala His Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys 115 120 125 Glu Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn 130 135 140 Ile Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Arg 145 150 155 160 Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala 165 170 175 Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val 180 185 190 Gly Gly 42 2253 DNA Escherichia coli gene (1)..(2253) Nucleotidic sequence coding for the quimeric protein in the plasmid pAZ2 42 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagat 180 agactcaaga tggacaaatt gaaactcaag gggatgagct atgcaatgtg cttgaatacc 240 tttgtgttga agaaagaagt ctccgaaacg cagcatggga caatactcat taaggttgag 300 tacaaagggg aagatgcacc ctgcaagatt cctttctcca cggaggatgg acaagggaaa 360 gctcacaatg gcagactgat cacagccaat ccagtggtga ccaagaagga ggagcctgtc 420 aacattgagg ctgaacctcc ttttggggaa agtaatatag taattggaat tggagacaaa 480 gccctgaaaa tcaactggta caggaaggga agctcgattg ggaagatgtt cgaggccact 540 gccagaggtg caaggcgcat ggccatcttg ggagacacag cctgggactt tggatctgtg 600 ggtggtgtga attcgatgaa ttcgatggac gtacctgctg aagttgcagg cgtagtcaaa 660 gaagttaaag ttaaagtcgg cgacaaaatc tctgaaggtg gtttgattgt cgtcgttgaa 720 gctgaaggca cggcagccgc tcctaaagcc gaagcggctg ccgccccggc gcaagaagcc 780 cctaaagctg ccgctcctgc tccgcaagcc gcgcaattcg gcggttctgc cgatgccgag 840 tacgacgtgg tcgtattggg tggcggtccc ggcggttact ccgctgcatt tgccgctgcc 900 gatgaaggct tgaaagtcgc catcgtcgaa cgttacaaaa ctttgggcgg cgtttgcctg 960 aacgtcggct gtatcccttc caaagccttg ttgcacaatg ccgccgttat cgacgaagtg 1020 cgccacttgg ctgccaacgg tatcaaatac cccgagccgg aactcgacat cgatatgctt 1080 cgcgcctaca aagacggcgt agtttcccgc ctcacgggcg gtttggcagg tatggcgaaa 1140 agccgtaaag tggacgttat ccaaggcgac gggcaattct tagatccgca ccacttggaa 1200 gtgtcgctga ctgccggcga cgcgtacgaa caggcagccc ctaccggcga gaaaaaaatc 1260 gttgccttca aaaactgtat cattgcagca ggcagccgcg taaccaaact gcctttcatt 1320 cctgaagatc cgcgcatcat cgattccagc ggcgcattgg ctctgaaaga agtaccgggc 1380 aaactgctga ttatcggcgg cggcattatc ggcctcgaga tgggtacggt ttacagcacg 1440 ctgggttcgc gtttggatgt ggttgaaatg atggacggcc tgatgcaagg cgcagaccgc 1500 gatttggtaa aagtatggca aaaacaaaac gaataccgtt ttgacaacat tatggtcaac 1560 accaaaaccg ttgcagttga gccgaaagaa gacggcgttt acgttacctt tgaaggcgcg 1620 aacgcgccta aagagccgca acgctacgat gccgtattgg ttgccgccgg ccgcgcgccc 1680 aacggcaaac tcatcagcgc ggaaaaagca ggcgttgccg taaccgatcg cggcttcatc 1740 gaagtggaca aacaaatgcg taccaatgtg ccgcacatct acgccatcgg cgacatcgtc 1800 ggtcagccga tgttggcgca caaagccgtt cacgaaggcc acgttgccgc cgaaaactgc 1860 gccggccaca aagcctactt cgacgcacgc gtgattccgg gcgttgccta cacttccccc 1920 gaagtggcgt gggtgggcga aaccgaactg tccgccaaag cctccggccg caaaatcacc 1980 aaagccaact tcccgtgggc ggcttccggc cgtgcgattg ccaacggttg cgacaagccg 2040 tttaccaagc tgatttttga tgccgaaacc ggccgcatca tcggcggcgg cattgtcggt 2100 ccgaacggtg gcgatatgat cggcgaagtc tgccttgcca tcgaaatggg ctgcgacgcg 2160 gcagacatcg gcaaaaccat ccacccgcac ccgggcgaat ccatcggtat ggcggcggaa 2220 gtggcattgg gtacttgtac cgacaaaaaa aaa 2253 43 747 PRT Escherichia coli CHAIN (1)..(747) Aminoacidic sequence of the PAZ2 43 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Leu Asp Arg Leu Lys Met Asp Lys Leu Lys Leu Lys Gly 50 55 60 Met Ser Tyr Ala Met Cys Leu Asn Thr Phe Val Leu Lys Lys Glu Val 65 70 75 80 Ser Glu Thr His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu 85 90 95 Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys 100 105 110 Ala His Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys 115 120 125 Glu Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn 130 135 140 Ile Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Arg 145 150 155 160 Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala 165 170 175 Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val 180 185 190 Gly Gly Val Asn Ser Met Asn Ser Met Asp Val Pro Ala Glu Val Ala 195 200 205 Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu 210 215 220 Gly Gly Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro 225 230 235 240 Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala 245 250 255 Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu 260 265 270 Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala 275 280 285 Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr 290 295 300 Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys 305 310 315 320 Ala Leu Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala 325 330 335 Ala Asn Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu 340 345 350 Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala 355 360 365 Gly Met Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln 370 375 380 Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala 385 390 395 400 Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys 405 410 415 Asn Cys Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile 420 425 430 Pro Glu Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys 435 440 445 Glu Val Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu 450 455 460 Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val 465 470 475 480 Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys 485 490 495 Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn 500 505 510 Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr 515 520 525 Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val 530 535 540 Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu 545 550 555 560 Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys 565 570 575 Gln Met Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val 580 585 590 Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala 595 600 605 Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile 610 615 620 Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr 625 630 635 640 Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe 645 650 655 Pro Trp Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro 660 665 670 Phe Thr Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly 675 680 685 Gly Ile Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu 690 695 700 Ala Ile Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His 705 710 715 720 Pro His Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 725 730 735 Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys 740 745 44 2256 DNA Escherichia coli gene (1)..(2256) Nucleotidic sequence coding for the quimeric protein in the plasmid pAZ3 44 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct 180 gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa 240 ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg 300 gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa 360 ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt 420 tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac 480 aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac 540 aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag 600 ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg 660 ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa 720 ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca 780 gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc 840 cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca 900 ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc 960 gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac 1020 ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac 1080 cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc 1140 gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta 1200 ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt 1260 gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac 1320 atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa 1380 ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt 1440 ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc 1500 aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg 1560 attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc 1620 atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt 1680 gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc 1740 ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct 1800 ccgcaaaaga aaaaaggatc cagactcaag atggacaaat tgaaactcaa ggggatgagc 1860 tatgcaatgt gcttgaatac ctttgtgttg aagaaagaag tctccgaaac gcagcatggg 1920 acaatactca ttaaggttga gtacaaaggg gaagatgcac cctgcaagat tcctttctcc 1980 acggaggatg gacaagggaa agctcacaat ggcagactga tcacagccaa tccagtggtg 2040 accaagaagg aggagcctgt caacattgag gctgaacctc cttttgggga aagtaatata 2100 gtaattggaa ttggagacaa agccctgaaa atcaactggt acaggaaggg aagctcgatt 2160 gggaagatgt tcgaggccac tgccagaggt gcaaggcgca tggccatctt gggagacaca 2220 gcctgggact ttggttcagt gggtggttaa ggatcc 2256 45 744 PRT Escherichia coli CHAIN (1)..(744) Aminoacidic sequence of the PAZ3 45 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val 50 55 60 Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 65 70 75 80 Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 85 90 95 Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 100 105 110 Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 115 120 125 Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 130 135 140 Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 145 150 155 160 Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu 165 170 175 Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn 180 185 190 Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala 195 200 205 Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met 210 215 220 Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu 225 230 235 240 Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu 245 250 255 Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys 260 265 270 Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 275 280 285 Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 290 295 300 Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 305 310 315 320 Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 325 330 335 Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 340 345 350 Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 355 360 365 Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 370 375 380 Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 385 390 395 400 Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala 405 410 415 Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met 420 425 430 Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln 435 440 445 Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu 450 455 460 Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly 465 470 475 480 Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu 485 490 495 Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp 500 505 510 Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 515 520 525 Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 530 535 540 Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 545 550 555 560 Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 565 570 575 Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys 580 585 590 Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Arg Leu Lys Met Asp 595 600 605 Lys Leu Lys Leu Lys Gly Met Ser Tyr Ala Met Cys Leu Asn Thr Phe 610 615 620 Val Leu Lys Lys Glu Val Ser Glu Thr His Gly Thr Ile Leu Ile Lys 625 630 635 640 Val Glu Tyr Lys Gly Glu Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr 645 650 655 Glu Asp Gly Gln Gly Lys Ala His Asn Gly Arg Leu Ile Thr Ala Asn 660 665 670 Pro Val Val Thr Lys Lys Glu Glu Pro Val Asn Ile Glu Ala Glu Pro 675 680 685 Pro Phe Gly Glu Ser Asn Ile Val Ile Gly Ile Gly Asp Lys Ala Leu 690 695 700 Lys Ile Asn Trp Tyr Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu 705 710 715 720 Ala Thr Ala Arg Gly Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala 725 730 735 Trp Asp Phe Gly Ser Val Gly Gly 740 46 426 DNA Escherichia coli gene (1)..(426) Nucleotidic sequence coding for the aminoacids 286 to 426 of the DEN-4 envelope protein 46 aaagtccgta tggagaaatt gagaatcaag ggaatgtcat acacgatgtg ttcaggaaag 60 ttttcaattg acaaagagat ggcagaaaca cagcatggga caacagtggt gaaagtcaag 120 tatgaaggtg ctggagctcc gtgtaaagtc cccatagaga taagagatgt aaacaaggaa 180 aaagtggttg ggcgtatcat ctcatccacc cctttggctg agaataccaa cagtgtaacc 240 aacatagaat tagaaccccc ctttggggac agctacatag tgataggtgt tggaaacagc 300 gcattaacac tccattggtt caggaaaggg agttccattg gcaagatgtt tgagtccaca 360 tacagaggtg caaaacgaat ggccattcta ggtgaaacag cttgggattt tggttccgtt 420 ggtgga 426 47 615 DNA Escherichia coli gene (1)..(615) Nucleotidic sequence coding for the quimeric protein in the plasmid pID1 47 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagac 180 aaagtgcgta tggagaaatt gagaatcaag ggaatgtcat acacgatgtg ttcaggaaag 240 ttttcaattg acaaagagat ggcagaaaca cagcatggga caacagtggt gaaagtcaag 300 tatgaaggtg ctggagctcc gtgtaaagtc cccatagaga taagagatgt aaacaaggaa 360 aaagtggttg ggcgtatcat ctcatccacc cctttggctg agaataccaa cagtgtaacc 420 aacatagaat tagaaccccc ctttggggac agctacatag tgataggtgt tggaaacagc 480 gcattaacac tccattggtt caggaaaggg agttccattg gcaagatgtt tgagtccaca 540 tacagaggtg caaaacgaat ggccattcta ggtgaaacag cttgggattt tggttccgtt 600 ggtggataag gatcc 615 48 192 PRT Escherichia coli CHAIN (1)..(192) Aminoacidic sequence of the PID1 48 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly Met Ser 50 55 60 Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met Ala Glu 65 70 75 80 Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly Ala Gly 85 90 95 Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys Glu Lys 100 105 110 Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn Thr Asn 115 120 125 Ser Val Thr Asn Ile Glu Leu Glu Arg Pro Leu Asp Ser Tyr Ile Val 130 135 140 Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg Lys Gly 145 150 155 160 Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala Lys Arg 165 170 175 Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val Gly Gly 180 185 190 49 2241 DNA Escherichia coli gene (1)..(2241) Nucleotidic sequence coding for the quimeric protein in the plasmid pID2 49 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagacaa agtccgtatg 180 gagaaattga gaatcaaggg aatgtcatac acgatgtgtt caggaaagtt ttcaattgac 240 aaagagatgg cagaaacaca gcatgggaca acagtggtga aagtcaagta tgaaggtgct 300 ggagctccgt gtaaagtccc catagagata agagatgtaa acaaggaaaa agtggttggg 360 cgtatcatct catccacccc tttggctgag aataccaaca gtgtaaccaa catagaatta 420 gaacccccct ttggggacag ctacatagtg ataggtgttg gaaacagcgc attaacactc 480 cattggttca ggaaagggag ttccattggc aagatgtttg agtccacata cagaggtgca 540 aaacgaatgg ccattctagg tgaaacagct tgggattttg gtagcgttgg tggactgaat 600 tcgatgaatt cgatggacgt acctgctgaa gttgcaggcg tagtcaaaga agttaaagtt 660 aaagtcggcg acaaaatctc tgaaggtggt ttgattgtcg tcgttgaagc tgaaggcacg 720 gcagccgctc ctaaagccga agcggctgcc gccccggcgc aagaagcccc taaagctgcc 780 gctcctgctc cgcaagccgc gcaattcggc ggttctgccg atgccgagta cgacgtggtc 840 gtattgggtg gcggtcccgg cggttactcc gctgcatttg ccgctgccga tgaaggcttg 900 aaagtcgcca tcgtcgaacg ttacaaaact ttgggcggcg tttgcctgaa cgtcggctgt 960 atcccttcca aagccttgtt gcacaatgcc gccgttatcg acgaagtgcg ccacttggct 1020 gccaacggta tcaaataccc cgagccggaa ctcgacatcg atatgcttcg cgcctacaaa 1080 gacggcgtag tttcccgcct cacgggcggt ttggcaggta tggcgaaaag ccgtaaagtg 1140 gacgttatcc aaggcgacgg gcaattctta gatccgcacc acttggaagt gtcgctgact 1200 gccggcgacg cgtacgaaca ggcagcccct accggcgaga aaaaaatcgt tgccttcaaa 1260 aactgtatca ttgcagcagg cagccgcgta accaaactgc ctttcattcc tgaagatccg 1320 cgcatcatcg attccagcgg cgcattggct ctgaaagaag taccgggcaa actgctgatt 1380 atcggcggcg gcattatcgg cctcgagatg ggtacggttt acagcacgct gggttcgcgt 1440 ttggatgtgg ttgaaatgat ggacggcctg atgcaaggcg cagaccgcga tttggtaaaa 1500 gtatggcaaa aacaaaacga ataccgtttt gacaacatta tggtcaacac caaaaccgtt 1560 gcagttgagc cgaaagaaga cggcgtttac gttacctttg aaggcgcgaa cgcgcctaaa 1620 gagccgcaac gctacgatgc cgtattggtt gccgccggcc gcgcgcccaa cggcaaactc 1680 atcagcgcgg aaaaagcagg cgttgccgta accgatcgcg gcttcatcga agtggacaaa 1740 caaatgcgta ccaatgtgcc gcacatctac gccatcggcg acatcgtcgg tcagccgatg 1800 ttggcgcaca aagccgttca cgaaggccac gttgccgccg aaaactgcgc cggccacaaa 1860 gcctacttcg acgcacgcgt gattccgggc gttgcctaca cttcccccga agtggcgtgg 1920 gtgggcgaaa ccgaactgtc cgccaaagcc tccggccgca aaatcaccaa agccaacttc 1980 ccgtgggcgg cttccggccg tgcgattgcc aacggttgcg acaagccgtt taccaagctg 2040 atttttgatg ccgaaaccgg ccgcatcatc ggcggcggca ttgtcggtcc gaacggtggc 2100 gatatgatcg gcgaagtctg ccttgccatc gaaatgggct gcgacgcggc agacatcggc 2160 aaaaccatcc acccgcaccc gggcgaatcc atcggtatgg cggcggaagt ggcattgggt 2220 acttgtaccg acaaaaaaaa a 2241 50 747 PRT Escherichia coli CHAIN (1)..(747) Aminoacidic sequence of the PID2 50 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Leu Asp Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly 50 55 60 Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met 65 70 75 80 Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly 85 90 95 Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys 100 105 110 Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn 115 120 125 Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Arg Pro Leu Asp Ser Tyr 130 135 140 Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg 145 150 155 160 Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala 165 170 175 Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val 180 185 190 Gly Gly Leu Asn Ser Met Asn Ser Met Asp Val Pro Ala Glu Val Ala 195 200 205 Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu 210 215 220 Gly Gly Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro 225 230 235 240 Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala 245 250 255 Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu 260 265 270 Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala 275 280 285 Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr 290 295 300 Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys 305 310 315 320 Ala Leu Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala 325 330 335 Ala Asn Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu 340 345 350 Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala 355 360 365 Gly Met Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln 370 375 380 Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala 385 390 395 400 Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys 405 410 415 Asn Cys Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile 420 425 430 Pro Glu Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys 435 440 445 Glu Val Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu 450 455 460 Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val 465 470 475 480 Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys 485 490 495 Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn 500 505 510 Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr 515 520 525 Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val 530 535 540 Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu 545 550 555 560 Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys 565 570 575 Gln Met Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val 580 585 590 Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala 595 600 605 Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile 610 615 620 Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr 625 630 635 640 Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe 645 650 655 Pro Trp Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro 660 665 670 Phe Thr Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly 675 680 685 Gly Ile Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu 690 695 700 Ala Ile Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His 705 710 715 720 Pro His Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 725 730 735 Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys 740 745 51 2256 DNA Escherichia coli gene (1)..(2256) Nucleotidic sequence coding for the quimeric protein in the plasmid pID3 51 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct 180 gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa 240 ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg 300 gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa 360 ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt 420 tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac 480 aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac 540 aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag 600 ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg 660 ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa 720 ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca 780 gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc 840 cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca 900 ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc 960 gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac 1020 ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac 1080 cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc 1140 gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta 1200 ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt 1260 gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac 1320 atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa 1380 ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt 1440 ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc 1500 aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg 1560 attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc 1620 atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt 1680 gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc 1740 ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct 1800 ccgcaaaaga aaaaaggatc caaagtgcgt atggagaaat tgagaatcaa gggaatgtca 1860 tacacgatgt gttcaggaaa gttttcaatt gacaaagaga tggcagaaac acagcatggg 1920 acaacagtgg tgaaagtcaa gtatgaaggt gctggagctc cgtgtaaagt ccccatagag 1980 ataagagatg taaacaagga aaaagtggtt gggcgtatca tctcatccac ccctttggct 2040 gagaatacca acagtgtaac caacatagaa ttagaacccc cctttgggga cagctacata 2100 gtgataggtg ttggaaacag cgcattaaca ctccattggt tcaggaaagg gagttccatt 2160 ggcaagatgt ttgagtccac atacagaggt gcaaaacgaa tggccattct aggtgaaaca 2220 gcttgggatt ttggttcggt tggtggctaa ggatcc 2256 52 744 PRT Escherichia coli CHAIN (1)..(744) Aminoacidic sequence of the PID3 52 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val 50 55 60 Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 65 70 75 80 Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 85 90 95 Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 100 105 110 Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 115 120 125 Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 130 135 140 Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 145 150 155 160 Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu 165 170 175 Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn 180 185 190 Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala 195 200 205 Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met 210 215 220 Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu 225 230 235 240 Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu 245 250 255 Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys 260 265 270 Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 275 280 285 Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 290 295 300 Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 305 310 315 320 Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 325 330 335 Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 340 345 350 Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 355 360 365 Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 370 375 380 Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 385 390 395 400 Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala 405 410 415 Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met 420 425 430 Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln 435 440 445 Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu 450 455 460 Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly 465 470 475 480 Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu 485 490 495 Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp 500 505 510 Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 515 520 525 Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 530 535 540 Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 545 550 555 560 Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 565 570 575 Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys 580 585 590 Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Lys Val Arg Met Glu 595 600 605 Lys Leu Arg Ile Lys Gly Met Ser Tyr Thr Met Cys Ser Gly Lys Phe 610 615 620 Ser Ile Asp Lys Glu Met Ala Glu Thr Gln His Gly Thr Thr Val Val 625 630 635 640 Lys Val Lys Tyr Glu Gly Ala Gly Ala Pro Cys Lys Val Pro Ile Glu 645 650 655 Ile Arg Asp Val Asn Lys Glu Lys Val Val Gly Arg Ile Ile Ser Ser 660 665 670 Thr Pro Leu Ala Glu Asn Thr Asn Ser Val Thr Asn Ile Glu Leu Glu 675 680 685 Arg Pro Leu Asp Ser Tyr Ile Val Ile Gly Val Gly Asn Ser Ala Leu 690 695 700 Thr Leu His Trp Phe Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu 705 710 715 720 Ser Thr Tyr Arg Gly Ala Lys Arg Met Ala Ile Leu Gly Glu Thr Ala 725 730 735 Trp Asp Phe Gly Ser Val Gly Gly 740 53 2694 DNA Escherichia coli gene (1)..(2694) Nucleotidic sequence coding for the quimeric protein in the plasmid pD4D2 53 atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg 60 aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg 120 ggcgacacta ttgctgtgga cgataccctg attactttgg atctagacaa agtccgtatg 180 gagaaattga gaatcaaggg aatgtcatac acgatgtgtt caggaaagtt ttcaattgac 240 aaagagatgg cagaaacaca gcatgggaca acagtggtga aagtcaagta tgaaggtgct 300 ggagctccgt gtaaagtccc catagagata agagatgtaa acaaggaaaa agtggttggg 360 cgtatcatct catccacccc tttggctgag aataccaaca gtgtaaccaa catagaatta 420 gaacccccct ttggggacag ctacatagtg ataggtgttg gaaacagcgc attaacactc 480 cattggttca ggaaagggag ttccattggc aagatgtttg agtccacata cagaggtgca 540 aaacgaatgg ccattctagg tgaaacagct tgggattttg gttccgttgg tggtcttcta 600 gaaatggacg tacctgctga agttgcaggc gtagtcaaag aagttaaagt taaagtcggc 660 gacaaaatct ctgaaggtgg tttgattgtc gtcgttgaag ctgaaggcac ggcagccgct 720 cctaaagccg aagcggctgc cgccccggcg caagaagccc ctaaagctgc cgctcctgct 780 ccgcaagccg cgcaattcgg cggttctgcc gatgccgagt acgacgtggt cgtattgggt 840 ggcggtcccg gcggttactc cgctgcattt gccgctgccg atgaaggctt gaaagtcgcc 900 atcgtcgaac gttacaaaac tttgggcggc gtttgcctga acgtcggctg tatcccttcc 960 aaagccttgt tgcacaatgc cgccgttatc gacgaagtgc gccacttggc tgccaacggt 1020 atcaaatacc ccgagccgga actcgacatc gatatgcttc gcgcctacaa agacggcgta 1080 gtttcccgcc tcacgggcgg tttggcaggt atggcgaaaa gccgtaaagt ggacgttatc 1140 caaggcgacg ggcaattctt agatccgcac cacttggaag tgtcgctgac tgccggcgac 1200 gcgtacgaac aggcagcccc taccggcgag aaaaaaatcg ttgccttcaa aaactgtatc 1260 attgcagcag gcagccgcgt aaccaaactg cctttcattc ctgaagatcc gcgcatcatc 1320 gattccagcg gcgcattggc tctgaaagaa gtaccgggca aactgctgat tatcggcggc 1380 ggcattatcg gcctcgagat gggtacggtt tacagcacgc tgggttcgcg tttggatgtg 1440 gttgaaatga tggacggcct gatgcaaggc gcagaccgcg atttggtaaa agtatggcaa 1500 aaacaaaacg aataccgttt tgacaacatt atggtcaaca ccaaaaccgt tgcagttgag 1560 ccgaaagaag acggcgttta cgttaccttt gaaggcgcga acgcgcctaa agagccgcaa 1620 cgctacgatg ccgtattggt tgccgccggc cgcgcgccca acggcaaact catcagcgcg 1680 gaaaaagcag gcgttgccgt aaccgatcgc ggcttcatcg aagtggacaa acaaatgcgt 1740 accaatgtgc cgcacatcta cgccatcggc gacatcgtcg gtcagccgat gttggcgcac 1800 aaagccgttc acgaaggcca cgttgccgcc gaaaactgcg ccggccacaa agcctacttc 1860 gacgcacgcg tgattccggg cgttgcctac acttcccccg aagtggcgtg ggtgggcgaa 1920 accgaactgt ccgccaaagc ctccggccgc aaaatcacca aagccaactt cccgtgggcg 1980 gcttccggcc gtgcgattgc caacggttgc gacaagccgt ttaccaagct gatttttgat 2040 gccgaaaccg gccgcatcat cggcggcggc attgtcggtc cgaacggtgg cgatatgatc 2100 ggcgaagtct gccttgccat cgaaatgggc tgcgacgcgg cagacatcgg caaaaccatc 2160 cacccgcacc cgaccttggg cgaatccatc ggtatggcgg cggaagtggc attgggtact 2220 tgtaccgacc tgcctccgca aaagaaaaaa ggatccgaca ggctgagaat ggacaaacta 2280 cagctcaaag gaatgtcata ctctatgtgt acaggaaagt ttaaaattgt gaaggaaata 2340 gcagaaacac aacatggaac aatagttatc agagtacaat atgaagggga cggctctcca 2400 tgtaagatcc cttttgagat aatggatttg gaaaaaagac acgtcttagg tcgcctgatt 2460 acagttaacc cgatcgtaac agaaaaagat agcccagtca acatagaagc agaacctcca 2520 ttcggagaca gctacatcat cataggagta gagccgggac aattgaaact caactggttt 2580 aagaaaggaa gttccatcgg ccaaatgttt gagacaacaa tgagaggagc gaagagaatg 2640 gccattttag gtgacacagc ctgggatttt gggtctctgg gtggttaagg atcc 2694 54 891 PRT Escherichia coli CHAIN (1)..(891) Aminoacidic sequence of the PD4D2 54 His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 1 5 10 15 Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 20 25 30 Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 35 40 45 Thr Leu Asp Leu Asp Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly 50 55 60 Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met 65 70 75 80 Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly 85 90 95 Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys 100 105 110 Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn 115 120 125 Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Arg Pro Leu Asp Ser Tyr 130 135 140 Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg 145 150 155 160 Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala 165 170 175 Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val 180 185 190 Gly Gly Leu Leu Glu Met Asn Ser Met Asp Val Pro Ala Glu Val Ala 195 200 205 Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu 210 215 220 Gly Gly Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro 225 230 235 240 Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala 245 250 255 Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu 260 265 270 Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala 275 280 285 Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr 290 295 300 Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys 305 310 315 320 Ala Leu Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala 325 330 335 Ala Asn Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu 340 345 350 Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala 355 360 365 Gly Met Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln 370 375 380 Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala 385 390 395 400 Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys 405 410 415 Asn Cys Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile 420 425 430 Pro Glu Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys 435 440 445 Glu Val Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu 450 455 460 Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val 465 470 475 480 Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys 485 490 495 Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn 500 505 510 Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr 515 520 525 Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val 530 535 540 Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu 545 550 555 560 Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys 565 570 575 Gln Met Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val 580 585 590 Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala 595 600 605 Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile 610 615 620 Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr 625 630 635 640 Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe 645 650 655 Pro Trp Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro 660 665 670 Phe Thr Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly 675 680 685 Gly Ile Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu 690 695 700 Ala Ile Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His 705 710 715 720 Pro His Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 725 730 735 Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Arg Leu Arg 740 745 750 Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly 755 760 765 Lys Phe Lys Ile Val Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile 770 775 780 Val Ile Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro 785 790 795 800 Phe Glu Ile Met Asp Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile 805 810 815 Thr Val Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu 820 825 830 Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro 835 840 845 Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln 850 855 860 Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala Ile Leu Gly 865 870 875 880 Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly Gly 885 890 

What is claimed is:
 1. Chimeric nucleotide chains comprising the combination of sequences corresponding to fragments of the E protein from Flavivirus, with sequences corresponding to the codifying gene for the mutated MDH protein, identified in the sequencing list as Sequence No.26, Sequence No.29 or with the fragment of these identified as Sequence No.23, which codify for chimeric proteins capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient, against the Flavivirus, when these proteins are present in a pharmaceutical preparation.
 2. Chimeric nucleotide chains according to claim 1, comprising the combination of one or several sequences that codify for the amino acids 286 to 426 of the E protein from Dengue viruses with the nucleotide sequence of the MDH protein, where these sequences are inserted and/or fused to the sequences identified in the sequencing list as Sequence No.26, Sequence No.29 or Sequence No.23, which codify for chimeric proteins capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient, serotype-specific against each serotype of Dengue virus.
 3. Chimeric nucleotide chains according to claim 1, comprising the nucleotide sequence that codifies for the amino acids 286 to 426 of the E protein from each serotype 1, 2, 3 and 4 of Dengue virus, fused to the 3′ end region that codifies for the first 45 amino acids of the MDH protein, identified as Sequence No.23.
 4. Chimeric nucleotide chains according to claim 1, comprising the nucleotide sequence that codifies for the amino acids 286 to 426 of the E protein from each serotype 1, 2, 3 and 4 of Dengue virus, fused to the 3′ end region that codifies for the amino acid 45 of the MDH protein, identified as Sequence No.26.
 5. Chimeric nucleotide chains according to claim 1, comprising the nucleotide sequence that codifies for the amino acids 286 to 426 of the E protein from each serotype 1, 2, 3 and 4 of Dengue virus, fused to the 3′ end region that codifies for the MDH protein, identified as Sequence No.29.
 6. Chimeric nucleotide chains according to claim 1, comprising the combination of the nucleotide sequences that codify for the amino acids 286 to 426 of the E protein from two different serotypes of Dengue virus, with the sequence of the MDH, where one of the sequences corresponding to the E protein is inserted in the region that codifies for the amino acid 45 of the MDH protein and the other is fused to the 3′ region of the same MDH sequence, identified as sequence No.29.
 7. Recombinant chimeric proteins characterized by being the expression products of the nucleotide chains of claim 1, and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient, serotype-specific against each serotype of Dengue virus, when these proteins are present in a pharmaceutical preparation.
 8. Chimeric recombinant proteins characterized by being the expression products of the nucleotide chains of claim 1, and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient, specific against the two homologous serotypes of Dengue virus.
 9. Chimeric nucleotide chains, identified essentially by the Sequence No.24, according to claim 1, comprising the combination of the sequences identified in the sequencing list as Sequence No.22 and Sequence No.23, which codifies for the chimeric protein PLL1.
 10. Chimeric protein PLL1, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.25, being the expression product of the Sequence No.24 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-2 virus, when PLL1 is present in a pharmaceutical preparation.
 11. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.27, comprising the combination of the sequences identified in the sequencing list as Sequence No.22 and Sequence No.26, which codifies for the chimeric protein PLL2.
 12. Chimeric protein PLL2, according to claim 1, characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.28, be the expression product of the Sequence No. 27 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-2 virus, when PLL2 is present in a pharmaceutical preparation.
 13. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.30, comprising the combination of the sequences identified in the sequencing list as Sequence No.22 and Sequence No.29, which codifies for the chimeric protein PLL3.
 14. Chimeric protein PLL3, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.31, being the expression product of the Sequence No.30 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-2 virus, when PLL3 is present in a pharmaceutical preparation.
 15. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.33, comprising the combination of the sequences identified in the sequencing list as Sequence No.23 and Sequence No.32, which codifies for the chimeric protein PLH1.
 16. Chimeric protein PLH1, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.34, being the expression product of the Sequence No.33 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-1 virus, when PLH1 is present in a pharmaceutical preparation.
 17. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.35, comprising the combination of the sequences identified in the sequencing list as Sequence No.26 and Sequence No.32, which codifies for the chimeric protein PLH2.
 18. Chimeric protein PLH2, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.36, being the expression product of the Sequence No.35 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-1 virus, when PLH2 is present in a pharmaceutical preparation.
 19. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.37, comprising the combination of the sequences identified in the sequencing list as Sequence No.29 and Sequence No.32, which codifies for the chimeric protein PLH3.
 20. Chimeric protein PLH3, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.38, being the expression product of the Sequence No.37 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-1 virus, when PLH3 is present in a pharmaceutical preparation.
 21. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.40, comprising the combination of the sequences identified in the sequencing list as Sequence No.23 and Sequence No.39, which codifies for the chimeric protein PAZ1.
 22. Chimeric protein PAZ1, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.41, being the expression product of the Sequence No.40 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-3 virus, when PAZ1 is present in a pharmaceutical preparation.
 23. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.42, comprising the combination of the sequences identified in the sequencing list as Sequence No.26 and Sequence No.39, which codifies for the chimeric protein PAZ2.
 24. Chimeric protein PAZ2, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.43, being the expression product of the Sequence No.42 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-3 virus, when PAZ2 is present in a pharmaceutical preparation.
 25. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.44, comprising the combination of the sequences identified in the sequencing list as Sequence No.29 and Sequence No.39, which codifies for the chimeric protein PAZ3.
 26. Chimeric protein PAZ3, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.45, being the expression product of the Sequence No.44 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-3 virus when, PAZ3 is present in a pharmaceutical preparation.
 27. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.47, comprising the combination of the sequences identified in the sequencing list as Sequence No.23 and Sequence No.46, which codifies for the chimeric protein PID1.
 28. Chimeric protein PID1, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.48, being the expression product of the Sequence No.47 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-4 virus, when PID1 is present in a pharmaceutical preparation.
 29. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.49, comprising the combination of the sequences identified in the sequencing list as Sequence No.26 and Sequence No.46, which codifies for the chimeric protein PID2.
 30. Chimeric protein PID2, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.50 being the expression product of the Sequence No.49 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-4 virus, when PID2 is present in a pharmaceutical preparation.
 31. Chimeric nucleotide chains according to claim 1 identified essentially as Sequence No.51, comprising the combination of the sequences identified in the sequencing list as Sequence No.29 and Sequence No.46, which codifies for the chimeric protein PID3.
 32. Chimeric protein PID3, according to claim 1 characterized by having essentially the amino acid sequence identified in the sequencing list as Sequence No.52 being the expression product of the Sequence No.51 and being capable of inducing a humoral immune response of neutralizing and protective antibodies in the recipient against the Dengue-4 virus, when PID3 is present in a pharmaceutical preparation.
 33. A pharmaceutical preparation capable of inducing in the recipient a protective immune response, specific against the Dengue virus, according to claim 1, characterized by having two or more of the chimeric proteins described in claim 1, and a pharmacological vehicle.
 34. A pharmaceutical preparation according to claim 33, characterized by being a preventive or therapeutic agent against the Dengue viruses, for oral, intramuscular, subcutaneous, mucosal or intravenous use.
 35. A diagnostic mean characterized by having one or several of the proteins described in claim 1, useful for the diagnosis and serotyping of the Dengue virus 